Kinesin heavy chain


DEVELOPMENTAL BIOLOGY

Kermit interacts with gαo, vang, and motor proteins in Drosophila planar cell polarity

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue - the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen Kermit, previously implicated in G protein and PCP signaling, was uncovered as a novel binding partner of Go. Through pull-down and genetic interaction studies, it was found that Kermit interacts with Go and another PCP component Vang (Strabismis), known to undergo intracellular relocalization during PCP establishment. It was further demonstrated that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI (Jaguar). The results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment (Lin, 2013).

At the top of the signaling hierarchy in PCP lies a G protein-coupled receptor Fz. The heterotrimeric Go protein emerged as an immediate transducer of Fz in Drosophila as well as other organisms. One of the mediators of Go signaling in PCP is the endocytic GTPase Rab5 required for the proper Fz internalization and relocalization. During PCP establishment, Fz concentrates at the distal apical position of wing epithelia. This study describes identification of Kermit as another transducer of Go in PCP. kermit downregulation suppresses the Gαo-overexpression phenotypes, and Gαo and kermit co-overexpression results in a prominent synergism in PCP malformations (Lin, 2013).

Kermit and its mammalian homolog GIPC, through their PDZ domain, are known to interact with a number of proteins in various organisms. Observations in Xenopus and mice indicated that Kermit/GIPC could interact with members of the Fz and RGS protein families -- Fz3, Fz7, and RGS19 (De Vries, 1998; Tan, 2001). Since Go also binds Fz and RGS proteins, it was hypothesized that a quaternary complex consisting of Fz, Go, Kermit, and RGS19 could form in Drosophila PCP, with Kermit as a potential organizer of these interactions. However, Drosophila Kermit was found not interact with Fz. Similarly, no binding between Kermit and the Drosophila RGS19 homolog could be seen. Thus Kermit is unlikely to act as a scaffold in Fz-Go signaling, and another mode of action of Kermit in transducing Go signal exists in PCP (Lin, 2013).

In a recent study using mouse genetics and cellular assays, a role of GIPC1 in regulating Vangl2 (a murine homolog of Drosophila Vang) intracellular trafficking has been revealed (Giese, 2012). In Drosophila PCP, Vang relocalizes to the site opposite to Fz at the proximal apical tip of wing epithelia. This study provides genetic evidence placing Vang downstream from Kermit in Drosophila PCP, suggesting that the Kermit-Vang connection is conserved from insects to mammals (Lin, 2013).

kermit expression is strongly upregulated in the developing wing during PCP establishment, and kermit overexpression induces strong PCP phenotypes (Djiane, 2010). In Xenopus, both up- and down-regulation of kermit lead to defective Fz3-dependent neural crest induction. It is thus surprising that Drosophila kermit loss-of-function alleles were homozygous viable and did not reveal PCP phenotypes. It is proposed that Kermit may regulate Drosophila PCP redundantly with some other PDZ domain-containing proteins, such as Scribble or Patj, which genetically interact with PCP components but on their own also produce only mild phenotypes; of those Scribble has been shown to interact with Vang both in Drosophila and mammals. In general, up to 75% of genes Drosophila are estimated to be phenotypically silent in loss-of-function due to redundancy, and the significance of gain-of-function analysis in discovery of novel important pathway components has been highlighted in a recent large-scale Drosophila-based assay. Kermit, based on the presented overexpression and genetic interaction studies, can thus be considered as an important regulator of Drosophila PCP (Lin, 2013).

A genetic and physical interaction between Kermit and the unconventional actin-based motor MyoVI has been described. This study confirmed that the dominant UAS-kermit PCP phenotypes critically depend on the MyoVI activity. MyoVI has been previously shown to mediate removal of endocytic vesicles away from the cell's periphery. The excessive activity of Kermit or MyoVI may thus result in removal of Vang-containing vesicles from the apical membrane, contributing to mislocalization of Vang and appearance of the PCP defects. In contrast, microtubule-based transport along the apical microtubule cables, polarized below the apical plasma membrane in wing epithelia, mediates the correct relocalizations of Fz and Vang in PCP. It is probable that a competition between the actin-based and microtubule-based motors may exist for the endocytic vesicles containing PCP components, and that excessive Kermit activity unbalances this competition in favor of the actin-based transport. Thus whether reduction in the levels of the microtubule-based transport system would further aggravate the dominant UAS-kermit PCP phenotypes was tested. And indeed, reduction in either the minus end-directed motor dynein or the plus end-directed motor kinesin significantly enhances the UAS-kermit effects (Lin, 2013).

The following model is proposed to collectively explain the results. It is speculated that endocytic vesicles containing PCP components can be transported in a planar manner, along the microtubule meshwork underlying the apical plasma membrane -- the mode of transport required for the proper apical relocalizations of these components. Alternatively, the vesicles can be trapped by the actin cables and transported away from the apical membrane, removing them from the active pool of PCP components. In the case of Vang, the choice between these decisions is regulated by the Kermit protein, which favors the actin-based transport (Lin, 2013).

These findings and model shed new light on the mechanisms of complex inter-regulations ensuring the robust epithelial polarization, likely conserved across the metazoans (Lin, 2013).

Pavarotti/MKLP1 regulates microtubule sliding and neurite outgrowth in Drosophila neurons

Kinesin-1 can slide microtubules against each other, providing the mechanical force required for initial neurite extension in Drosophila neurons. This sliding is only observed in young neurons actively forming neurites and is dramatically downregulated in older neurons. The downregulation is not caused by the global shutdown of kinesin-1, as the ability of kinesin-1 to transport membrane organelles is not diminished in mature neurons, suggesting that microtubule sliding is regulated by a dedicated mechanism. This study has identified the "mitotic" kinesin-6 Pavarotti (Pav-KLP) as an inhibitor of kinesin-1-driven microtubule sliding. Depletion of Pav-KLP in neurons strongly stimulated the sliding of long microtubules and neurite outgrowth, while its ectopic overexpression in the cytoplasm blocked both of these processes. Furthermore, postmitotic depletion of Pav-KLP in Drosophila neurons in vivo reduced embryonic and larval viability, with only a few animals surviving to the third instar larval stage. A detailed examination of motor neurons in the surviving larvae revealed the overextension of axons and mistargeting of neuromuscular junctions, resulting in uncoordinated locomotion. Taken together, these results identify a new role for Pav-KLP as a negative regulator of kinesin-1-driven neurite formation. These data suggest an important parallel between long microtubule-microtubule sliding in anaphase B and sliding of interphase microtubules during neurite formation (Del Castillo, 2014).

Previous work showed that microtubule sliding by kinesin-1 drives initial neurite outgrowth in Drosophila neurons and that sliding is downregulated as neurons mature. This paper, has demonstrated that the 'mitotic' kinesin Pav-KLP functions as a negative regulator of interphase microtubule sliding both in Drosophila S2 cells and in Drosophila neurons. Knockdown of Pav-KLP stimulated microtubule sliding, producing longer axons, while overexpression of Pav-KLP inhibited both sliding and neurite outgrowth. Increased length of axons after Pav-KLP depletion was also observed in vivo in Drosophila. Therefore, Pav-KLP attenuates neurite outgrowth by downregulation of kinesin-1-powered microtubule-microtubule sliding (Del Castillo, 2014).

Pav-KLP and its orthologs (members of the kinesin-6 family) were originally identified as essential components for central spindle assembly and cleavage furrow formation. Pav-KLP depletion induces defects in morphology of the mitotic spindle at telophase and failure to recruit contractile ring components. However, it has been demonstrated that CHO1/MKLP1, the mammalian ortholog of Pav-KLP, has an additional function in neurodevelopment. CHO1/MKLP1 plays a role in establishing dendrite identity in differentiated neurons. Depletion of CHO1/MKLP1 induces progressive loss of dendrites. It has concluded that CHO1/MKLP1 organizes microtubules in dendrites by transporting short minus-end-distal microtubule fragments into the dendrites. More recent work has revisited the role of CHO1/MKLP1 in developing neurons and suggested that CHO1/MKLP1 can regulate neurite outgrowth. Depletion of CHO1/MKLP1 increased transport of short microtubule fragments. The current data are in agreement with the idea that Pav-KLP regulates formation of neurites. However, the mechanisms reported in this study are clearly different from the results obtained by the mammalian studies in two significant aspects. First, this study has shown that the reorganization of microtubules required for neurite formation is driven by kinesin-1. Second, the current visualization technique clearly demonstrates that microtubules in developing Drosophila neurons are moved as long polymers. It is possible that the differences between the results and the mammalian study can be explained by different models (Drosophila versus mammalian neurons). A more attractive idea is that similar mechanisms work in both systems, but further work is required to understand the role of kinesin-1 in neurite outgrowth in mammalian neurons (Del Castillo, 2014).

Interestingly, work by several groups has shown that proteins that function together with kinesin-6 in the cytokinesis pathway could also regulate neuronal morphogenesis. For example, Tumbleweed or Ect2/Pebble/RhoGEF depletion increases the extent of neurite outgrowth, suggesting that Tumbleweed and RhoGEF control neurite outgrowth through actin reorganization. However, the current results demonstrate that the primary regulator of neurite outgrowth is kinesin-6 family member Pav-KLP, the essential partner of Pebble and Tumbleweed. Furthermore, the effect of Pav-KLP on process formation is independent of actin or small GTPases (although more subtle effects of Tumbleweed or Ect2 on the actin cytoskeleton in developing neurons cannot be completely excluded). Indeed, a recent work concluded that an actin-signaling pathway regulated by the Centralspindlin complex controls protrusive activity required for directional neuronal migration (Del Castillo, 2014).

The original idea that mitotic motors regulate cytoplasmic microtubules in neurons suggested that microtubule arrays in neurons are established by mechanisms that are analogous to those that organize the mitotic spindle. Supporting this idea, it was demonstrated that inhibition of other mitotic motors, e.g., kinesin-5, affected the axon length Advancing this concept, this paper proposes that Pav-KLP/kinesin-6 directly regulates cytoplasmic microtubule arrangement by crosslinking them. It has been shown that loss-of-function mutations on ZEN-4/MKLP1, the C. elegans form of Pav-KLP, produce longer spindles, suggesting that kinesin-6 motors inhibit sliding of microtubules against each other during anaphase B. If this is indeed the case, the current results suggest an important functional similarity between the molecular mechanisms of cell division and process formation in neurons. While anaphase B is driven in part by microtubule-microtubule sliding powered by bipolar kinesin-5 and negatively regulated by kinesin-6 (mammalian MKLP1/C. elegans Zen-4/Drosophila Pav-KLP), the initial formation of neurites requires microtubule-microtubule sliding by kinesin-1 and, similar to anaphase B, is negatively regulated by kinesin-6. Thus, kinesin-6 motors together with other components of the Centralspindlin complex can function as general brakes of microtubule-microtubule sliding during both cell division and postmitotic neurite formation (Del Castillo, 2014).

The fact that microtubule sliding is inhibited by Pav-KLP in mature, but not young, neurons suggests that Pav-KLP itself is temporally regulated. One potential mechanism that could affect the ability of Pav-KLP (MKLP-1) to regulate microtubule sliding is Pav-KLP phosphorylation. Phosphorylation of Ser710 in MKLP-1 (Ser743 in Drosophila Pav-KLP) has been shown to promote its binding to protein 14-3-3, preventing MKLP-1 from clustering on microtubules. Future studies using phosphomimetic variants of Pav-KLP may help to test this mechanism (Del Castillo, 2014).

Kinesin-1-powered microtubule sliding initiates axonal regeneration in Drosophila cultured neurons

Understanding the mechanism underlying axon regeneration is of great practical importance to develop therapeutic treatment for traumatic brain and spinal cord injuries. Dramatic cytoskeleton reorganization occurs at the injury site, and microtubules have been implicated in the regeneration process. Previous studies have demonstrated that microtubule sliding by conventional kinesin (Kinesin-1) is required for initiation of neurite outgrowth in Drosophila embryonic neurons, and that sliding is developmentally down-regulated when neurite outgrowth is completed. This study reports that mechanical axotomy of Drosophila neurons in culture triggers axonal regeneration and regrowth. Regenerating neurons contain actively sliding microtubules; this sliding, like sliding during initial neurite outgrowth, is driven by Kinesin-1 and is required for axonal regeneration. The injury induces a fast spike of calcium, and depolymerization of microtubules near the injury site, and subsequent formation of local new microtubule arrays with mixed polarity. These events are required for reactivation of microtubule sliding at the initial stages of regeneration. Furthermore, c-Jun N-terminal kinase (JNK) pathway promotes regeneration by enhancing microtubule sliding in injured mature neurons (Lu, 2015).

Oocyte

Microtubules and the Kinesin heavy chain (the force-generating component of the plus end-directed microtubule motor Kinesin I) are required for the localization of oskar mRNA to the posterior pole of the Drosophila oocyte, an essential step in the determination of the anteroposterior axis. The Kinesin heavy chain is also required for the posterior localization of Dynein, and for all cytoplasmic movements within the oocyte. Furthermore, the KHC localizes transiently to the posterior pole in an oskar mRNA-independent manner. Surprisingly, cytoplasmic streaming still occurs in kinesin light chain null mutants, and both oskar mRNA and Dynein localize to the posterior pole. Thus, the Kinesin heavy chain can function independently of the light chain in the oocyte, indicating that it associates with its cargoes by a novel mechanism (Palacios, 2002).

To determine whether kinesin functions in the same step of oskar mRNA localization as the other proteins required for this process, the distribution of the RNA in germline clones of a null allele of the Kinesin heavy chain, Khc27 were compared to barentsz, staufen and mago nashi mutants. Although no oskar mRNA reaches the posterior of the stage 9 oocyte in Khc27, there is a clear difference in the distribution of the mRNA from that observed in the other mutants, such as barentsz. In the latter, oskar mRNA remains tightly localized at the anterior cortex, whereas it is found throughout the anterior half of the oocyte in Khc27 mutant clones. Fluorescent in situ hybridization was performed to examine the distribution of oskar mRNA in the Khc mutant in more detail, using confocal microscopy. This reveals an anterior-to-posterior gradient of mRNA with an enrichment along the lateral cortex. Consistent with this, antibody staining for Staufen protein shows a distribution identical to oskar mRNA. These results suggest that the Khc mutant blocks oskar mRNA localization after it has been released from the anterior cortex, whereas all of the other factors are required for this release (Palacios, 2002).

Several lines of evidence have suggested that the light chain is essential for the function of conventional kinesin in vivo. Mutants in the Drosophila light chain are lethal, and produce the same block in fast axonal transport as mutants in the kinesin heavy chain, leading to axonal swelling and progressive posterior paralysis. Mutants in one of the mouse KLC genes also interfere with the function of the heavy chain, by causing its aberrant accumulation near the cis-Golgi. One proposed role for the light chain is to regulate the activity of the motor domain. The light chain inhibits the ATPase activity of the motor in vitro, and co-transfection experiments in tissue culture cells have demonstrated that it represses the binding of the heavy chain to microtubules. Since the phenotypes of Klc mutants indicate that it also plays a positive role in kinesin function, it may inhibit motor activity in the absence of cargo, but activate it upon cargo binding. A second essential function of the light chain is to couple the heavy chain to its cargoes. In all known cases specific cargo interactions with kinesin are mediated by the light chain (Palacios, 2002).

Drosophila KHC is required in the oocyte for the posterior localization of oskar mRNA, the posterior localization of DHC and for cytoplasmic streaming. In light of the results of this study, it is very surprising that the light chain is dispensable for the three functions of kinesin in the Drosophila female germline. One trivial explanation is that there is a second light chain gene in Drosophila, but this seems highly unlikely for several reasons: (1) the protein is not redundant in the nervous system, since a strong axonal transport phenotype is observed in Klc mutants; (2) there is only one light chain gene in the 'complete' Drosophila genome sequence (63% sequence identity to human kinesin light chain 1), and all of the light chain cDNAs in the extensive Drosophila EST collections correspond with this gene; (3) the 'complete' genome sequence of another Dipteran insect, the mosquito Anopheles gambiae, also contains only a single Klc gene. Although it is possible that there is a second light chain gene in the small region of each genome that has not been sequenced, it seems very improbable that this would be the case in both organisms. Thus, these results strongly suggest that the kinesin heavy chain can function without a light chain in the oocyte, and that it must therefore interact with its cargo or cargoes in some other way (Palacios, 2002).

Although there is no precedent for light chain independent activities of the KHC in higher eukaryotes, the distantly related kinesin heavy chains of fungi, such as Neurospora crassa, function without any associated light chains. Mutagenesis studies on the N. crassa kinesin have identified a putative cargo-binding domain in the tail, and this region has been conserved in animal KHCs. It may therefore represent an alternative cargo-binding domain that could account for the light chain independence of the KHC functions in the oocyte. Interestingly, the glutamate receptor interacting protein, GRIP1, has recently been shown to bind to this region of the mouse KHC. GRIP1 has been proposed to target kinesin to dendrites, and it is not yet known whether it functions as a cargo adaptor, or plays a role in light chain independent transport (Palacios, 2002).

Twenty years ago it was suggested that the vigorous ooplasmic streaming and the cytoplasmic movements in the nurse cells in stage 10b egg chambers are independent processes. The results of the current study demonstrate that this is indeed the case, not only at stage 10b, but also earlier in oogenesis, since ooplasmic streaming is completely abolished in Khc mutant egg chambers, whereas the cytoplasmic movements in the nurse cells and from the nurse cells into the oocyte are unaffected. It is unclear how kinesin creates these cytoplasmic flows in the oocyte. Given its role in vesicle transport in other systems, an attractive model is that it transports some organelle or vesicle along microtubules, and that this then generates flows in the surrounding cytoplasm, because of its viscosity. It seems unlikely that kinesin is directly transporting any of the particles or vesicles that have been visualised, since these particles move at speeds of about 0.1 µm/second at stage 9, which is significantly slower than other reported kinesin-dependent transport processes. This suggests that kinesin generates streaming by transporting some other organelle or vesicle more rapidly along the microtubules (Palacios, 2002).

The nature of the cytoplasmic flows in the oocyte is variable and temporally regulated. The ooplasmic streaming at stage 9 is slow and uncoordinated, whereas the movements at stage 10b are faster and unidirectional, and resemble those of a 'washing machine'. As both types of ooplasmic streaming are completely abolished in Khc mutants, these differences cannot be due to the motor protein. The type of streaming probably depends, at least in part, on the organization of the microtubule cytoskeleton, which changes completely at the beginning of stage 10b, but kinesin may also have distinct cargoes at the two stages, which could influence the strength of the cytoplasmic flows (Palacios, 2002).

In an attempt to understand the mechanism for oskar mRNA transport to the posterior, the movement of a GFP-Staufen fusion protein was analyzed in living oocytes. Although this fusion protein localizes to the posterior with oskar mRNA and rescues the oskar mRNA localization defect of a staufen null mutant, movements that unambiguously correspond to posterior transport have not been resolved. One possible explanation for this failure is that most of the fluorescent GFP-Staufen particles do not contain oskar mRNA, which is expressed at much lower levels than the fusion protein. Thus, the relevant oskar mRNA/GFP-Staufen complexes may be too rare or too weakly fluorescent to follow in time-lapse films. Although it was not possible to determine how GFP-Staufen reaches the posterior, the results do reveal several important features of this process that are relevant to the discussion of the models for the mechanism of oskar mRNA localization (Palacios, 2002).

One model proposes that cytoplasmic flows circulate oskar mRNA around the oocyte, so that it can then be efficiently trapped at the posterior by a pre-localized cortical anchor. Indeed, this mechanism would account for the failure to detect any directed transport of GFP-Staufen to the posterior pole. The observation that the KHC is required for all cytoplasmic flows in the oocyte also supports this model, since it provides an explanation for why the KHC is required to localize oskar mRNA. However, several other considerations make this mechanism unlikely. (1) The cytoplasmic flows are much weaker at the posterior of the oocyte than elsewhere, presumably because there are fewer microtubules in this region, and many oocytes show little or no cytoplasmic movement near the posterior pole. It is therefore hard to imagine how cytoplasmic flows could efficiently deliver the mRNA to a posterior anchor. (2) The hypothetical anchor would have to localize to the posterior before oskar mRNA and in an oskar mRNA independent manner, and no proteins that meet these criteria have been identified so far. Indeed, the only proteins that fulfil the second criterion are the KHC and the components of the dynein/dynactin complex. (3)oskar mRNA localizes to the center of the oocyte in mutants that alter the organization of the microtubule cytoskeleton, such as gurken, pka and par-1, and it is hard to reconcile this with trapping by a cortical anchor, since there is no plasma membrane or cortical cytoskeleton in this region. The localization of oskar mRNA still correlates with the position of microtubule plus ends in these mutants, because Kin-ßGal forms a dot in the center of the oocyte with the mRNA, and this is more consistent with the model in which oskar mRNA is transported along microtubules towards the posterior pole. Finally, the KHC accumulates at the posterior during the stages when oskar mRNA and DHC are localized, strongly suggesting that KHC plays a direct role in transporting them there (Palacios, 2002).

Another model for oskar mRNA localization proposes that the KHC functions to transport the RNA away from the minus ends of the microtubules at the anterior and lateral cortex towards the plus ends in the interior of the oocyte, and that the lack of microtubules at the posterior somehow allows the mRNA to accumulate at this pole. Two aspects of the data do not fit this cortical exclusion model. (1) No oskar mRNA or Staufen was seen at the posterior of the oocyte in Khc germline clones, regardless of whether fluorescent or wholemount in situ hybridization or antibody staining was performed. This observation seems incompatible with a model in which kinesin removes oskar mRNA from the anterior and lateral cortex, but is not required for its localization to the posterior pole. (2) The demonstration that endogenous kinesin localizes to the posterior cortex, like kinesin-ßGal, provides further evidence that the plus ends of the microtubules are enriched in this region, and strongly suggests that kinesin mediates transport to this pole. These localizations are not visible until stage 9, however, which is when oskar mRNA starts to accumulate at the posterior. Thus, conflicting results can be resolved by proposing that the plus ends lie in the middle of the oocyte at stage 8, when a kinesin-dependent accumulation of oskar mRNA in the central dot is seen, and that microtubules are only recruited to the posterior at stage 9, coincident with the onset of oskar mRNA localization (Palacios, 2002).

In light of the posterior localization of endogenous kinesin, it is thought most likely that this motor does transport oskar mRNA to the posterior of the oocyte, even though this movement has not yet been seen. The link between the KHC and the oskar mRNA localization complex need not be direct, however. The KHC probably transports something else to the posterior of the oocyte, in addition to oskar mRNA and dynein. This is thought to be so because mutants that abolish either oskar mRNA localization (such as staufen and barentsz) or DHC localization (Dhc64C6-6/Dhc64C6-12) have no effect on the posterior localization of the KHC, even though the motor activity of the KHC is thought to require binding to a cargo. The KHC is also required for cytoplasmic streaming, and presumably induces these flows by moving a large structure, such as a vesicle or organelle, along microtubules. This structure should therefore accumulate at the posterior of the oocyte during stage 9, because this is where the microtubule plus ends and the KHC itself localize. Thus, oskar mRNA and dynein could reach the posterior at stage 9 by hitch-hiking on the large cargo that drives streaming. This proposal is consistent with several other observations: (1) the fact that cytoplasmic streaming, oskar mRNA localization and dynein localization all share the very unusual property of being light chain independent suggests that they all depend on a single KHC-mediated transport process, which could be the transport of the cargo that induces streaming to the posterior; (2) it has been shown in a number of other systems that plus and minus end directed microtubule motors, such as kinesin and dynein, are found on the same organelles; (3) if dynein and oskar mRNA interact with the kinesin cargo independently of each other, this would explain why both their posterior localizations require the KHC, but do not require each other, and finally, (4) there is already evidence that links oskar mRNA localization with vesicle trafficking, since mutants in rab11, a small GTPase implicated in the regulation of endocytic vesicle recycling, disrupt the posterior localization of oskar mRNA. Furthermore, Rab11 itself localizes to the posterior of the oocyte. The effect of Rab11 on oskar mRNA localization may be indirect, however, since these mutants also disrupt the organization of the microtubule cytoskeleton (Palacios, 2002).

It is unclear why dynein localizes to the posterior, but one possibility is that it is needed to recycle kinesin to the minus ends of the microtubules, so that kinesin can mediate another round of posterior localization. The only known phenotype of the Dhc64C mutants that specifically disrupt the posterior localization of DHC is a reduction in the rate of cytoplasmic streaming, and this may due to the gradual depletion of the pool of KHC available for transport. However, this localization may be important for recycling dynein away from the minus ends of microtubules, so that dynein can mediate further rounds of minus end-directed transport (Palacios, 2002).

If the hitch-hiking model for oskar mRNA localization is correct, Staufen, Barentsz, Mago nashi and Y14 would be required to couple the mRNA to the vesicle or organelle that is transported by kinesin. In this context, it is interesting to note that mammalian Staufen homologs have been shown to associate with the endoplasmic reticulum. The localization of Vg1 mRNA to the vegetal pole of Xenopus oocytes requires the RNA-binding protein VERA/Vg1 RBP, which co-fractionates with markers for the endoplasmic reticulum, and this has led to the suggestion that Vg1 mRNA is transported in association with ER vesicles. Thus, hitchhiking on vesicles may represent a general mechanism for mRNA transport (Palacios, 2002).

In the Drosophila oocyte, microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. mRNA localization and nuclear positioning at mid-oogenesis is shown to depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. It is concluded that the microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole (Januschke, 2002).

The localization of bcd mRNA in the oocyte occurs in multiple steps. Several of these involve active transport along microtubules. bcd mRNA coassembles into particles with Exuperantia (Exu) in the nurse cells and in the oocyte. This complex is essential for the correct localization of bcd to the anterior cortex in a microtubule-dependent manner. During mid-oogenesis, bcd maintenance at the anterior cortex is dependent on Swallow (Swa). This protein harbors a putative double-strand-RNA binding motif and a coiled-coil domain, which interacts with the Dynein light chain (Dlc-1). Swa has been proposed to act as an adaptor between bcd mRNA and the Dynein motor. Swa itself localizes to the anterior cortex of stage-10 oocytes, and this localization requires the coiled-coil domain, suggesting that polar transport of Swa and its cargo, bcd mRNA, occurs in a Dynein-dependent manner. The observation that bcd mRNA is delocalized in oocytes overexpressing p50 Dynamitin (Dmn), a component of the Dynactin complex provides further support for this suggestion (Januschke, 2002).

Surprisingly, in khc mutant oocytes, bcd mRNA is not tightly concentrated to the anterior cortex but is diffusely spread out in a wide cortical ring that expands toward the posterior. Thus, correct bcd localization depends not only on minus end-directed, but also on plus end-directed, motors. Kinesin I might be directly involved in anchoring of bcd mRNA to the anterior cortex. Alternatively, Kinesin I might be required for efficient Dynein-dependent transport of bcd. The observation that Dynein is mislocalized in khc mutant oocytes supports the latter hypothesis. Dhc fails to accumulate at the posterior pole of khc mutant oocytes and instead is enriched at the anterior cortex. Thus, Kinesin I appears to be necessary to relocate Dynein to the posterior pole after it has moved together with its cargo to the anterior pole. This would allow for renewed rounds of cargo loading and transport to the anterior cortex. Without sustained posterior-to-anterior transport, the bcd mRNA/adaptor complexes might become delocalized by diffusion. This scenario indicates that sustained transport could be an alternative to an independent anchorage step (Januschke, 2002).

In contrast to what was detected with bcd, no dual motor requirement has been detected for osk localization to the posterior, which is Kinesin I dependent. osk mRNA is clearly localized and translated when Dynein function is impaired. However, several features of the phenotypes produced by Dynamitin overexpression suggest that Dynein function is not completely abolished. Thus, it cannot be strictly ruled out that low levels of Dynein are required for efficient osk transport to the posterior pole. Indeed, the mechanism of osk localization is more complex than that of bcd localization. osk localization occurs through a series of distinct steps first to the anterior, then to the middle, and finally to the posterior pole of the oocyte (Januschke, 2002).

The correct positioning of the oocyte nucleus requires two different anchoring processes: one to the lateral cortex and a second to the anterior cortex. The former might be a prerequisite for nuclear movement from the posterior to the anterior pole. The latter occurs after completion of the movement. While Kinesin I appears to be dispensable for nuclear movement, the role of Dynein remains to be clarified. The Dynein-Dynactin complex is essential for nuclear migration in many cell types, from yeast to vertebrates. Overexpression of Dmn, though, seems not to interfere with correct positioning of the nucleus at early stages. Since nuclear migration is targeted toward MT minus ends, like bcd mRNA localization, it is assumed that it requires Dynein and it is suggested that, in the Dmn overexpression experiment, residual Dynein function is present at early stages, which allows nuclear migration (Januschke, 2002).

While the question of migration needs further analysis, both motors are clearly required for nuclear anchorage. Impairment of Dynein leads to nuclear detachment from both the anterior and lateral cortex. Kinesin I, however, is only required for maintaining the nucleus at the anterior cortex. The fact that both anchoring processes fail when Dmn is overexpressed indicates that Dynein fulfils a complex function in nuclear positioning. Two Dynein-Dynactin pools are present in the oocyte: the posterior pool, which is microtubule dependent and maintained by Kinesin I-dependent transport, and the perinuclear pool, whose maintenance is independent of MTs and MT motor activity. The perinuclear Dynein-Dynactin pool appears to be involved in organizing a MT cage around the nucleus, and this cage is likely to be necessary for the attachment of the nucleus to the lateral cortex. The mislocalization of the nucleus in Kinesin mutants might be explained in a similar way as the mislocalization of bcd mRNA. Cortical Dynein activity might be required during some stage after the nucleus has reached the anterior pole. During this period what appears to be anchorage would be the result of sustained minus end-directed movement. To maintain this movement, Dynein is supplied from the posterior pool, which constantly has to be replenished by Kinesin-dependent transport (Januschke, 2002).

The nuclear MT scaffold might not only be important for nuclear migration and nuclear anchoring. It harbors centrosomal components such as Centrin, which probably contribute to the formation of the MT scaffold but might also influence the MT network of the entire oocyte. Due to these properties, the nucleus is likely to have a central role in polarizing the Drosophila oocyte. During migration, it might contribute to the overall anterior-posterior repolarization of the oocyte MT network, which is required to establish the anterior and posterior cortical domains. After migration, MTs emanating from the asymmetrically positioned nucleus are likely to polarize the transport of grk mRNA and Grk protein, which establishes the anterodorsal cortical domain (Januschke, 2002).

grk mRNA is produced by both the nurse cells and the oocyte nucleus. After nuclear migration, grk mRNA accumulates briefly along the anterior margin of the oocyte, before it concentrates in a perinuclear position. The anterior localization of grk is not affected when Dynein function is reduced or if Kinesin I function is completely abolished. However, both motors are required to transport grk to the nucleus. It is suggested that grk mRNA is transported toward the minus ends of MTs, which emanate from the nucleus. This would explain the Dynein requirement for grk transport to the nucleus. The role of Kinesin I in anterodorsal grk transport might again reflect the need to retrieve the Dynein motors for renewed cargo loading, as suggested for bcd and the oocyte nucleus (Januschke, 2002).

This model has to assume, however, that Dynein-Dynactin complexes carrying different cargos can distinguish between distinct populations of MTs: Dynein-Dynactin complexes loaded with bcd mRNA should be transported to and remain at anterior cortex, while those loaded with grk mRNA should be subject to a second transport step toward the nucleus. Deletions within the grk 3'UTR allow anterior localization of grk mRNA but prevent its transport to the nucleus. This suggests that specific factors distinguish anterior and anterodorsal transport of grk. The heterogeneous nuclear RNA binding protein (hnRNP) Squid plays a central role in this process. It regulates both grk localization and translation and binds directly to the grk 3'UTR. Squid protein, like grk, appears to be transiently localized along the anterior cortex during the transition from stage 7 to stage 8 (Januschke, 2002).

grk mRNA, though mislocalized, is frequently translated when Kinesin I or Dynein motor activities are impaired. Since grk mRNA is found around the anterior cortex in those cases, Grk secretion should occur around the entire circumference of the oocyte instead of being restricted to the dorsal side. Secreted Grk induces dorsal follicle cell fates. Thus, ectopic secretion should lead to the formation of dorsalized eggs as in squid and fs(1)K10 mutants in which grk mRNA is also mislocalized. However, impaired MT motor activity leads to ventralized eggs and thus to reduced Grk signaling. An analysis of Grk distribution in oocytes shows that, in contrast to wild-type or squid and fs(1)K10, Grk protein is not closely associated with grk mRNA and fails to reach the plasma membrane. Thus, polar transport of Grk protein and exocytosis requires Dynein and Kinesin I activity. This is not surprising, since both motors have been shown to be involved in Golgi dynamics in higher eukaryotes and it has been shown that vesicular trafficking from the Golgi to the plasma membrane requires Kinesin activity (Januschke, 2002).

Interestingly, no requirement has been detected for the two motors in earlier Grk signaling, which induces posterior follicle cells and prevents the formation of a second micropyle at the posterior pole. In the case of Dynamitin overexpression, this might be due once more to residual levels of Dynein function. In the case of Kinesin I, it is assumed that Grk secretion is only impaired, but not entirely blocked. The phenotypic series of grk mutations suggests that minute amounts of secreted Grk are sufficient to induce posterior follicle cells (Januschke, 2002).

Opposite polarity motors can interact with the same cargo in two fundamentally different ways. They can function in an opposition mode, like Myosin V and Kinesin II in the migration of Xenopus melanophores, or in a coordination mode, like Dynein and Kinesin in the motion of lipid droplets in the Drosophila embryo. In the opposition mode, the two motors produce opposing forces on a single cargo. Inactivation of the minus end-directed motor leads to a delocalization of the cargo to the plus end, whereas inactivation of the plus end-directed motor leads to a delocalization of the cargo to the minus end. In the coordination mode, the motors are not competing with each other. For example, when plus end motors are active, minus end motors are turned off, and vice versa. Inactivation of either of the two motors leads to the delocalization of the cargo to the same side. According to this scheme, Dynein and Kinesin I act in a coordination mode during transport of bcd and grk mRNAs, and the same might be true for their role in the positioning of the oocyte nucleus. The observation that Dynein accumulation at the posterior pole depends on Kinesin I suggests that both motors are associated with the same vesicle or macromolecular complex, as has been proposed for axonal transport in Drosophila. Dynein has to be inactive during Kinesin I-dependent transport to the posterior pole but then has to be activated again for renewed cargo loading and transport to the anterior cortex. If this recycling model holds true for the described polar transport processes in the oocyte, it will be challenging to find those factors that regulate motor activity and cargo loading in successive transport cycles (Januschke, 2002).

Despite their opposing polarity, Dynein and kinesin motors may cooperate in vivo. In Drosophila circumstantial evidence suggests that dynein acts in the localization of determinants and signaling factors during oogenesis. However, the pleiotropic requirement for dynein throughout development has made it difficult to establish its specific role. Dynein function in the oocyte has been examined by disrupting motor activity through temporally restricted expression of the dynactin subunit, dynamitin. The results indicate that dynein is required for several processes that impact patterning; such processes include localization of bicoid (bcd) and gurken (grk) mRNAs and anchoring of the oocyte nucleus to the cell cortex. Surprisingly, dynein function is sensitive to reduction in kinesin levels, and germ line clones lacking kinesin show defects in dorsal follicle cell fate, grk mRNA localization, and nuclear attachment that are similar to those resulting from the loss of dynein. Significantly, dynein and dynactin localization is perturbed in these animals. Conversely, kinesin localization also depends on dynein activity. It is concluded that dynein is required for nuclear anchoring and localization of cellular determinants during oogenesis. Strikingly, mutations in the kinesin motor also disrupt these processes and perturb dynein and dynactin localization. These results indicate that the activity of the two motors is interdependent and suggest a model in which kinesin affects patterning indirectly through its role in the localization and recycling of dynein (Duncan, 2002).

In order to investigate the effects of targeted disruption of dynein activity, heat shock-inducible (hsDmn) and Gal4-responsive (UAS-Dmn) transgenic lines were created. The hsDmn transgene permits tight temporal control of misexpression, whereas UAS-Dmn allows spatially restricted transcription when coupled with the appropriate Gal4 drivers. After mapping the insert position, hsDmn flies were examined for the ability to induce Dmn expression by probing immunoblots with polyclonal antisera against the Drosophila protein. A single band migrating at 45 kDa, close to the predicted size of the endogenous protein, was detected in untreated control flies. This band was present at approximately 5- to 10-fold higher levels in animals that had been heat shocked for 60 min. A time course of induction shows that elevated Dmn levels are present 15 min after heat shock and persist for at least 6 hr. To test whether Dmn overexpression perturbs the stability or localization (or both) of the dynein/dynactin complex in vivo, egg chambers were stained with antisera against Dmn, Gl (the largest subunit of dynactin), and the dynein intermediate chain (Cdic). In the wild-type, Dmn preferentially accumulates in the oocyte during early oogenesis and shows both perinuclear and cortical staining through stage 8. By stage 9 Dmn is enriched in a crescent at the posterior cortex as well as in lateral regions. Overall, this distribution mimics that of Gl and the dynein intermediate and heavy chains. Within 60 min of hsDmn induction, high levels of Dmn were detected throughout the oocyte, nurse cells and follicle cells. In contrast, Gl and Cdic staining was undetectable in stage 9/10 oocytes and was strongly reduced at earlier stages, demonstrating that Dmn overexpression disrupts the localization of the dynein/dynactin complex (Duncan, 2002).

One explanation for the strong genetic interaction observed between kinesin and Dmn overexpression could be that kinesin is required to transport dynein toward microtubule plus ends. This would allow individual dynein complexes to be reused for multiple rounds of minus end-directed motion. A reduction in kinesin levels may compromise this recycling and decrease the pool of available dynein; it would thus affect dynein's ability to translocate cargo toward microtubule minus ends. This model provides a mechanistic basis for why processes that involve dynein, such as nuclear attachment and grk RNA localization, could be severely impacted in oocytes lacking Khc. It is also consistent with the observation that in Khc mutant egg chambers the dynein/dynactin complex is not localized to the lateral cortex of the oocyte after stage 8. Significantly, localization of grk transcript and protein are relatively unaffected prior to stage 8, when defects in dynein/dynactin localization first become apparent (Duncan, 2002).

In this context, it is interesting that the distribution of Khc in the oocyte resembles that of dynein and dynactin components; i.e., it is enriched at the cortex and the perinuclear region, where microtubule minus ends are expected to be most abundant. Such a pattern is consistent with a role for kinesin in recycling dynein from the cortex, similar to its proposed function in transporting osk mRNA, but raises the paradoxical question of how kinesin localization is established. After hsDmn induction cortical staining for Khc is reduced, suggesting that Khc localization is in turn dependent on dynein activity. Transport of kinesin to the cortex could occur as a result of a direct physical interaction between the two motors. Alternatively, kinesin and dynein could bind common cargoes or adaptor proteins; this would be analogous to the situation in the embryo, where dynein and a so-far-unidentified plus-end motor both associate with individual lipid droplets. Transport of the particles and the associated motors could occur in either direction if the activity of the opposite polarity motors is appropriately regulated. Interaction with a common intermediate anchored to the posterior cortex could also explain why kinesin, dynein, and dynactin colocalize in this region. The recent finding that dynein-associated structures move rapidly along microtubules in both directions in Dictyostelium suggests that motor recycling may be a common mechanism for enhancing optimal utilization of a limited pool of these mechanochemical enzymes (Duncan, 2002).

The results indicate a role for dynein in grk transcript localization. The fact that grk mRNA cannot be detected in late-stage oocytes 1-6 hr after Dmn induction argues that dynein could be required for both the transport and anchoring of grk message. When microtubules are depolymerized, grk mRNA forms aggregates on the oocyte nuclear lamina, suggesting that this represents a site where it is anchored. It might therefore be expected that if dynein functions exclusively in transport, inhibition of its activity would cause an increase in the perinuclear concentration of grk mRNA. Furthermore, transcripts that were already at the cortex should not have been disrupted. In oocytes assayed 1 or 6 hr after Dmn induction, grk message was absent from the nuclear periphery and the cortex, irrespective of where the nucleus was positioned. However, 12 hr after Dmn induction, grk mRNA localization to the nucleus had partially recovered. Interestingly, when the oocyte nucleus was incorrectly positioned along the A/P axis but remained cortically attached, grk transcript was also detected at the cortex. This argues that nuclear position and proximity to the cortex are primary determinants of grk localization. It is notable that grk message is insensitive to Dmn overexpresssion and the absence of kinesin in earlier-stage egg chambers, when it may be transported by a diffusion-based mechanism and is known to accumulate even in the absence of microtubules or microfilaments. Localization of bcd message at the anterior of the oocyte is also highly susceptible to Dmn misexpression. Although the results cannot distinguish between inhibition of transport or anchoring of the mRNA, other data argue that dynein is likely to be involved in both of these aspects. Resolution of this issue may require direct observation of RNA localization in live egg chambers after hsDmn induction (Duncan, 2002).

In contrast to the dramatic effect of Dmn overexpression on grk and bcd transcripts, osk mRNA distribution is altered in a more subtle fashion. The increased level of osk mRNA in the cytoplasm after Dmn overexpression is consistent with the proposal that osk transcript binds to cortex throughout the oocyte and that kinesin transports it toward the interior in the anterior and lateral regions. Accordingly, dynein may contribute to osk localization by transporting transcripts toward the cortex or maintaining them there (Duncan, 2002).

The requirement for dynein activity in positioning the oocyte nucleus at the anterior cortex could reflect a role in nuclear anchoring alone or in both nuclear migration and anchoring. Misplaced nuclei are found in stage 10 egg chambers dissected 1 hr after heat shock even though nuclear migration would have occurred 13-25 hr earlier (at stage 7/8). This clearly shows that reduction of dynein activity disrupts nuclear anchoring through a mechanism that is still unclear. One possibility is that sustained activity of perinuclear dynein (acting on microtubules oriented with minus ends toward the cortex) is required to maintain nuclear position. Alternatively, cortically localized dynein may have to be continually active to keep the nucleus 'reeled in' through a subset of microtubules that have the opposite orientation. In either case, the nucleus would be predicted to fall away from the cortex in the absence of dynein activity. With respect to nuclear migration, there is considerable evidence that dynein motors power such a process in fungi. The data do not permit a firm conclusion as to whether dynein also performs this role in the oocyte. Although severely ventralized eggs were obtained 40 hr after hsDmn expression, suggesting a failure of nuclear migration, this could also result from defects in anchoring after migration because of perdurance of excess Dmn (Duncan, 2002).

Compared to oocytes in which dynein activity has been disrupted, those lacking kinesin show a higher frequency of nuclear-positioning defects. One explanation could be that kinesin function is completely abolished in Khc null clones, whereas residual dynein activity remains after hsDmn induction. Alternatively, it is conceivable that kinesin is the primary motor involved in nuclear positioning and that dynein plays an accessory role. In either event, the similarity in nuclear localization defects is consistent with a model in which the function of the two motors is linked. Dynein- and kinesin-related motors also act cooperatively to bring about nuclear migration in S. cerevisiae. Deletion of either of the kinesin-related proteins Kip2p and Kip3p or the dynein heavy chain results in nuclear migration defects. Epistatic analysis suggests that Kip2p acts cooperatively with dynein, whereas Kip3p may affect nuclear migration through an independent pathway involving Kar9p. Similarly, in Aspergillus, where nuclear migration is primarily thought to be dynein mediated, it has recently been shown that kinesin mutations affect nuclear movement and distribution in the hyphae. It is concluded that both dynein and kinesin are required for nuclear anchoring and localization of cellular determinants during oogenesis. The subcellular localization of dynein and dynactin is perturbed in kinesin mutants, and kinesin distribution is affected by Dmn misexpression. The interdependence of the two motors suggests a model in which kinesin affects patterning by localizing and recycling dynein and thus maximizing its utilization (Duncan, 2002 and references therein).

Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture

Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. This study used Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. Streaming can be used to detect subtle changes in Kinesin activity, and the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, estimates were established of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, it is suggested that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA (Ganguly, 2012).

The quantification of cytoplasmic streaming outlined in this study relates for the first time cellular changes affecting Kinesin activity and the large-scale properties of flows. The finding of a relation between motor activity and cytoplasmic streaming holds not only for mid-oogenesis (stage 9), but also for the fast unidirectional movement of the ooplasm at later stages, as it was also observed that flows at stage 11 are slower in pat1, encoding a protein that is required for Kinesin-1 to transport cargo and to maximize its motility (Loiseau, 2010), mutants than in WT flies. The results also show that the local velocity of streaming is a functional of the MT concentration, MT orientation, and the density of active Kinesin (Ganguly, 2012).

One presumed function of streaming is facilitating the distribution of cytoplasmic components that do not diffuse fast enough for proper cellular activity. In Drosophila oocytes, flows are not essential for localization of nanos mRNA at late-oogenesis, but they enhance its translocation to the oocyte posterior. Similarly, streaming at mid-oogenesis is not essential for the localization of oskar mRNA. However, the data suggest that motor-dependent flows could facilitate the localization of molecules by aiding the movement in the right direction of the cargo or cargo/motor complexes that detach from the MT cytoskeleton. The fact that streaming may be an essential variable when considering transport may be relevant to a range of process occurring in cells. The model framework introduced in this paper allows for a quantitative understanding of the time scales of motor driven transport, and its structure is applicable to any system with motor driven transport (Ganguly, 2012).

The findings of correlations between cytoplasmic streaming features and MT network architecture may allow the noninvasive, straightforward detection of streaming to be used to predict changes in cytoskeleton organization in vivo. This correlation is particularly interesting regarding tumorigenesis, where changes to the cytoskeleton occur, and quantified cytoplasmic streaming may thus serve as a diagnostic tool (Ganguly, 2012).

Dynein associates with oskar mRNPs and is required for their efficient net plus-end localization in Drosophila oocytes

In order for eukaryotic cells to function properly, they must establish polarity. The Drosophila oocyte uses mRNA localization to establish polarity and hence provides a genetically tractable model in which to study this process. The spatial restriction of oskar mRNA and its subsequent protein product is necessary for embryonic patterning. The localization of oskar mRNA requires microtubules and microtubule-based motor proteins. Null mutants in Kinesin heavy chain (Khc), the motor subunit of the plus end-directed Kinesin-1, result in oskar mRNA delocalization. Although the majority of oskar particles are non-motile in khc nulls, a small fraction of particles display active motility. Thus, a motor other than Kinesin-1 could conceivably also participate in oskar mRNA localization. This study shows that Dynein heavy chain (Dhc), the motor subunit of the minus end-directed Dynein complex, extensively co-localizes with Khc and oskar mRNA. In addition, immunoprecipitation of the Dynein complex specifically co-precipitated oskar mRNA and Khc. Lastly, germline-specific depletion of Dhc resulted in oskar mRNA and Khc delocalization. These results therefore suggest that efficient posterior localization of oskar mRNA requires the concerted activities of both Dynein and Kinesin-1 (Sanghavi, 2013).

Kinesin acts antagonistically to Dynein to maintain nuclear position within postmitotic Drosophila photoreceptor neurons

How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. The Dynactin complex (a regulator of the microtubule motor protein Dynein) has been shown to be required to maintain the position of the nucleus within post-mitotic Drosophila photoreceptor neurons. Multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. It has been found that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function (Whited, 2004).

The Dynactin complex is an assembly of 11 different subunits that functions as an activator of Dynein, serving as an adaptor for cargo and enhancing motor processivity. The Dynactin subunit Glued couples Dynactin to Dynein by binding to the Dynein intermediate chain (Dic, encoded by short wing). Overexpression of a truncated form of Glued that binds to Dic but cannot associate with the rest of the Dynactin complex acts as a powerful inhibitor of Dynein and Dynactin function. Overexpression of the Dynactin subunit Dynamitin disrupts Dynactin complex assembly and also inhibits Dynactin function. Biochemical studies have shown that the Dynactin complex also contains Capping Protein, a heterodimer composed of the Capping Protein alpha (Cpa) and Capping Protein beta (Cpb) subunits. Although best known for capping the barbed ends of filaments of actin, Capping Protein also associates with filaments of the actin-related Arp1 protein, which is a central element of the Dynactin complex (Whited, 2004 and references therein).

Patterning of the adult compound eye of Drosophila initiates during the third instar phase of larval life, and mutations in the Dynactin subunit Glued strongly disrupt eye development. Normally the nuclei of differentiating photoreceptors occupy apical regions of the eye disc. In animals heterozygous for the dominant-negative Glued allele Glued1, many photoreceptor nuclei have been shown to accumulate within basal regions of the eye disc. The effect of Glued1 on photoreceptor development was characterized using an antibody recognizing photoreceptor cell surfaces. In wild type, the region of the differentiating photoreceptor neuron containing the nucleus remained in the retina, while the photoreceptor axon extended through the optic stalk into the brain. However, in Glued1 animals, while photoreceptors still extended axons into the brain, the region of the photoreceptor containing the nucleus often appeared to leave the retina and travel through the optic stalk into the brain. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in Glued1 mutants (Whited, 2004).

To further establish that Glued1 defects reflected disruptions in Dynactin function, two other approaches were used to disrupt the Dynactin complex. Drosophila Dynamitin, which also inhibits Dynactin function in flies, was overexpressed in photoreceptor neurons. Loss-of-function mutations in the Dynactin subunit Cpb were examined by generating animals whose visual systems contained homozygous mutant clones of the cpb strong loss-of-function mutation cpbM143. In these cpbM143 mosaic animals, the nuclear regions of many photoreceptors were observed in the optic stalk and brain (Whited, 2004).

To confirm that the cpbM143 mutant photoreceptor defect was due to a loss of cpb function, an additional strong loss-of-function cpb allele, cpbF44, was isolated from an EMS mutagenesis and a chromosomal deficiency uncovering the cpb locus, Df(2L)E.2, was obtained. When animals contained homozygous mutant clones of cpbF44 cells or homozygous mutant clones of Df(2L)E.2, a similar movement of photoreceptor nuclear regions toward the brain was observed. cpb/Df(2L)E.2 animals did not survive to third instar, preventing the classic genetic demonstration that these cpb alleles behaved as strong loss-of-function mutations. Fortunately, it was found that the [pYES-ß] genomic transgene, which contains the CPB coding region, was able to rescue the lethality of cpb/Df(2L)E.2 animals, but did not rescue the previously described cpb bristle defect. This suggested that [pYES-ß] was a partially functional rescue construct that could be used to examine the visual systems of otherwise cpb/Df(2L)E.2 animals. It was found that [pYES-ß];cpbM143/Df(2L)E.2 animals display a photoreceptor defect similar to that of other cpb mutants, consistent with nuclear mispositioning resulting from the loss of cpb function. It was further confirmed that the defect was due to the loss of cpb function by successfully rescuing the cpbM143/Df(2L)E.2 photoreceptor defects (as well as the cpb bristle defects) by expression of a wild-type Cpb cDNA under the control of a heterologous promoter. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in cpb mutants (Whited, 2004).

The bifunctional nature of Cpb, which associates with filaments of actin as well as filaments of Arp1, means that loss of Cpb also increases filamentous actin levels (Hopmann, 2003). Nonetheless, previous studies have shown that increases in filamentous actin alone, such as those observed in hypomorphic cpb alleles or in actup mutants, do not cause photoreceptor nuclear mispositioning. Together with the Glued1 and Dynamitin data, the cpb observations yield a consistent picture that alterations in Dynactin subunits cause mispositioning of photoreceptor cell bodies and nuclei, and indicate that Dynactin, and not just the Glued subunit, has an important role in photoreceptor development (Whited, 2004).

The mispositioning of photoreceptor nuclei in Dynactin mutants raised the question of whether these disruptions reflect altered positioning of the nucleus within the photoreceptor or simply migration of the entire photoreceptor. To address this question, single photoreceptors were labeled in wild type and in Glued1 mutants. Wild-type photoreceptors exhibit a highly polarized morphology in which the region of the photoreceptor containing the nucleus lies in the apical region of the eye disc and an axon extends basally into the brain. Glued1 mutant photoreceptors whose nuclei have entered the optic stalk had highly altered morphologies, with both leading and trailing processes extending from the regions of the cell where the misplaced nucleus was located. Leading and trailing processes of misplaced Glued1 photoreceptors were quantified, considering only those with no other labeled cells or processes nearby. Of these 13 neurons, 12 had clearly detectable leading and trailing processes. The leading process (axon) extended into the target region and the trailing process extended back into the eye disc. These data demonstrate that inhibition of Dynactin function dramatically alters the position of the nucleus within the photoreceptor (Whited, 2004).

The Dynactin complex also controls the pattern of mitoses within the Drosophila retina. To determine whether nuclear mispositioning is a secondary consequence of the earlier mitotic requirement for Dynactin, the effects of specifically inhibiting the Dynactin complex in postmitotic photoreceptors was examined. Conditional inhibition of Dynactin function can be achieved through inducible expression of a truncated, dominant-negative form Glued (GluedDN) that resembles the protein product of Glued1. GluedDN was expressed under the control of the postmitotic photoreceptor-specific Glass 38-1 promoter, which initiates expression in the photoreceptors only after their axons have entered the brain. Expression of GluedDN under the control of Glass 38-1 caused photoreceptor nuclei to move into the optic stalk. Overexpression of Dynamitin under the control of Glass 38-1 caused similar photoreceptor nuclear positioning defects. These data demonstrate that Dynactin is required postmitotically in photoreceptors to maintain nuclear position and that the disruptions in nuclear positioning observed are not simply a secondary consequence of mitotic defects (Whited, 2004).

The displacement of photoreceptor nuclei from apical regions of the eye disc toward more basal regions could reflect an overall disruption in apical/basal polarity of the eye disc. The apical/basal polarity of developing photoreceptors was assessed by examining the distribution of the Drosophila ß-catenin Armadillo and the PDZ-domain-containing protein PATJ. Armadillo localizes to the zonula adherens separating the apical and basolateral membrane domains of developing photoreceptors, while PATJ localizes to the apical membrane domain. In wild-type eye discs, Armadillo is concentrated just beneath the apical tips of the developing photoreceptors. In Glued1 animals Armadillo was still present near apical regions of the eye disc, even in areas completely devoid of apical photoreceptor nuclei. Thus, this marker of apical/basal polarity was retained even when photoreceptor nuclei moved basally. Similar results were obtained when Glued1 mutants were visualized in cross-section using both Armadillo and PATJ. Apical localization of PATJ and Armadillo were observed in Glued1 and the relative apical/basal ordering of these markers was maintained. These data suggest that the alterations in photoreceptor morphology are not caused by a loss of apical/basal polarity within the developing photoreceptors (Whited, 2004).

Dynactin has important functions in the organization of the microtubule cytoskeleton in many systems. The microtubule cytoskeleton of developing photoreceptors is highly polarized, with microtubule minus ends concentrated apical to the nucleus as detected using antisera recognizing gamma-tubulin. A similar apical focus is observed when using the fusion protein Nod:LacZ, which often co-localizes with microtubule minus ends. The relatively ubiquitous expression of gamma-tubulin in the retina complicated the analysis of gamma-tubulin localization when retinal patterning was disrupted. Therefore, the effect of Glued on factors associated with the microtubule cytoskeleton was examined by expressing Nod:LacZ specifically in postmitotic photoreceptors. In animals expressing GluedDN in postmitotic photoreceptors as well as in Glued1 mutants, Nod:LacZ was no longer exclusively concentrated in apical regions of photoreceptors, but rather spread into the photoreceptor axons. Thus, while the overall apical/basal polarity of the photoreceptors was not disrupted in Glued mutants, the spatial organization of the microtubule cytoskeleton-associated factor Nod:LacZ was affected (Whited, 2004).

Dynactin activates the microtubule motor Dynein, and strong loss-of-function mutations in dynein intermediate chain (dic) are dominant enhancers of the rough eye phenotype of Glued1 mutants. Since Dynein and Dynactin may play multiple roles together during eye development, the effect of a reduction in dic gene dosage upon photoreceptor nuclear positioning was examined in Glued1 animals. A twofold reduction in dic gene dosage caused a further decrease in the number of photoreceptor nuclei in apical regions of Glued1 mutant eye discs. This did not reflect a simple reduction in the number of photoreceptors generated; large numbers of photoreceptor nuclei were crowded at the base of the eye disc and entered the optic stalk in both animals. Thus, a larger fraction of photoreceptor nuclei left apical positions when the level of dic gene activity was reduced, consistent with Dynein and Dynactin acting together in this process (Whited, 2004).

To identify additional factors that interact with Dynactin to control nuclear positioning, a genetic screen was performed to identify genes that dominantly enhanced or suppressed the Glued1 external eye phenotype. From a collection of approximately 1800 stocks containing transposon-induced lethal mutations, several stocks were identified that had no dominant effect on eye development in a wild-type background, but were dominant enhancers or suppressors of Glued1. Two dominant suppressors of Glued1, khck13219 and khck13314, were alleles of kinesin heavy chain (khc), which encodes a subunit of the plus-end directed microtubule motor kinesin. The interaction with Glued1 was further confirmed using the null allele khc8. Examination of developing eye discs demonstrated that a twofold reduction of khc gene dosage greatly increased the number of photoreceptor nuclei present in apical regions of Glued1 mutant eye discs. This suggested that khc acts antagonistically to Glued in photoreceptor nuclear positioning (Whited, 2004).

To determine whether khc mutations interacted with Glued1 in postmitotic photoreceptors, khc gene dosage was reduced in animals expressing dominant-negative Glued under the control of the postmitotic Glass38-1 promoter. Wild-type animals (n >50 hemispheres) or animals containing the dominant-negative Glued transgene without the Glass 38-1 promoter never contained photoreceptor nuclei within their optic stalks. Glass38-1:GluedDN animals contained an average of 11±1 photoreceptor nuclei within the optic stalk. However, Glass38-1:GluedDN animals heterozygous for either khck13314 or khc8 showed a significant reduction in the number of photoreceptor nuclei in the optic stalk. Thus, a twofold reduction in khc gene dosage suppressed the effects of postmitotic expression of dominant-negative Glued, consistent with Glued and khc acting antagonistically within differentiated photoreceptors to regulate nuclear positioning (Whited, 2004).

The interaction between Glued and khc in other photoreceptors was studied by examining the Bolwig organ, a cluster of 12 photosensitive neurons that differentiate during embryonic development and extend axons into the brain. By second and third instar larval stages, Bolwig photoreceptor nuclei are located near the anterior tip of the larva and their axons extend over the eye/antennal disc into the brain, a distance of >200 µm. In wild-type second instar animals, photoreceptor neuron differentiation has not yet begun in the eye disc and no neuronal nuclei are present there. However, when GluedDN was expressed in postmitotic Bolwig photoreceptors, their nuclei appeared on the surface of the eye/antennal disc. Thus, as in the photoreceptors of the adult eye, expression of GluedDN in Bolwig photoreceptors caused their nuclei to be positioned closer to their axon termini; in many cases, the Bolwig nuclei were over 150 µm closer than normal to their axon terminals in the brain (Whited, 2004).

The interaction between Glued and khc in Bolwig photoreceptors was assessed by counting the number of Bolwig nuclei on the surface of the eye/antennal disc. While wild-type and UAS:GluedDN animals had no neuronal nuclei in this region, Glass38-1:GluedDN animals contained 7±1. A reduction of khc gene dosage in Glass38-1:GluedDN; khck13314/+ and Glass38-1:GluedDN; khc8/+ animals significantly reduced this to 4±1 and 3±1, respectively. These data further support the functional antagonism of Glued and khc in photoreceptor nuclear positioning (Whited, 2004).

Control of RNP motility and localization by a splicing-dependent structure in oskar mRNA

oskar RNA localization to the posterior pole of the Drosophila melanogaster oocyte requires splicing of the first intron and the exon junction complex (EJC) core proteins. The functional link between splicing, EJC deposition and oskar localization has been unclear. This study demonstrates that the EJC associates with oskar mRNA upon splicing in vitro and that Drosophila EJC deposition is constitutive and conserved. In vivo analysis reveals that splicing creates the spliced oskar localization element (SOLE), whose structural integrity is crucial for ribonucleoprotein motility and localization in the oocyte. Splicing thus has a dual role in oskar mRNA localization: assembling the SOLE and depositing the EJC required for mRNA transport. The SOLE complements the EJC in formation of a functional unit that, together with the oskar 3' UTR, maintains proper kinesin-based motility of oskar mRNPs and posterior mRNA targeting (Ghosh, 2012).

mRNA localization is an evolutionarily conserved process allowing spatial and temporal restriction of protein synthesis to specific sites within cells. Active transport of mRNAs involves recognition of cis-acting sequence elements in the transcript by trans-acting factors and formation of ribonucleoprotein (RNP) particles, for which translocation to their final destination is mediated by motor proteins and the cytoskeleton. In Drosophila, oskar (osk) localization to the posterior pole of the oocyte depends on microtubules (MTs) and kinesin heavy chain (KHC). The identity of the cis element(s) that mediate oskar mRNA posterior localization is largely unknown. Although a previous study indicated that posterior localization signals reside in the oskar 3′ untranslated region (UTR), subsequent analysis revealed that the 3′ UTR is not sufficient to target an mRNA to the posterior pole. This indicated that other features of oskar RNA have a crucial role in the localization process (Ghosh, 2012).

Consistent with this, splicing of oskar at the position of the first intron is essential for posterior localization of the mRNA (Hachet, 2004). In addition, Y14, Mago nashi (Mago), eIF4AIII and Barentsz proteins are required for oskar mRNA transport in the oocyte. Notably, the vertebrate homologs of Drosophila Y14, Mago, eIF4AIII and Barentsz constitute the core components of the EJC and have been shown to associate with mRNAs, approximately 20-24 nucleotides (nt) upstream of exon-exon junctions, upon splicing. Although the requirements of splicing and the EJC core proteins for oskar mRNA localization indicate that nuclear events have a role in determining the assembly of localization-competent oskar mRNPs, the splicing-dependent association of the Drosophila EJC proteins with mRNAs, including oskar, has not been demonstrated (Ghosh, 2012).

To gain insight into the roles of splicing and the EJC in mRNA transport, this study combined biochemical, genetic and live-imaging approaches. It was shown that the Drosophila EJC is assembled on mRNAs upon splicing in vitro. Splicing of the first intron in oskar results in EJC deposition and formation of the SOLE, which has a predicted stem-loop structure. Mutational analysis of the SOLE shows that its proximal stem is required for proper oskar RNP motility and, therefore, for mRNA localization at the posterior pole of the oocyte. Taken together, these results shed light on the significance of splicing at the position of the first intron in oskar RNA and highlight the functional interconnection between the SOLE and the juxtaposed EJC (Ghosh, 2012).

This study shows that the EJC is deposited on ftz and oskar mRNAs as a consequence of splicing in embryo nuclear extract and that it binds at the same position relative to exon-exon junctions as in mammals. Hence, EJC assembly seems to be a constitutive and conserved process among metazoans (Ghosh, 2012).

The discovery of the bipartite SOLE provides an explanation for the observation that splicing of oskar RNA at the first intron is essential for localization of the mRNA (Hachet, 2004). The SOLE is formed by the joining of the 18 and 10 intron-proximal nucleotides of the first and second exons, respectively, upon splicing. However, the role of splicing is not simply to join these exonic sequences: mRNAs produced from oskΔi(1) or oskΔi(1,2,3) transgenes, which do not require splicing for formation of the 28-nt SOLE, are mislocalized (Hachet, 2004). All four EJC core proteins are required for oskar localization, and EJC deposition on mRNAs ~20-24 nt upstream of the exon-exon junction is splicing dependent; hence, a second important function of splicing is to load the EJC. Thus, EJC assembly and SOLE formation upon splicing are closely linked, both in time and space, with the EJC bound immediately adjacent to the SOLE (Ghosh, 2012).

Indeed, the relative configurations of the EJC deposition site and the SOLE seem to be important, as oskΔi(1) mRNA - which contains the 28 contiguous SOLE nucleotides and so should bear EJC complexes on exons 2 and 3 after splicing - fails to localize. Furthermore, the transgenic oskar RNAs contain a vector-derived intron at their 5′ end; hence, after splicing in vivo, mRNAs produced from oskΔi(1) and the intronless oskΔi(1,2,3) transgenes should have an EJC bound ~635 nt upstream of the SOLE, and yet neither oskΔi(1) nor oskΔi(1,2,3) mRNA is localized (Ghosh, 2012).

The relationship between EJC binding and the SOLE is unclear. The experiments indicate that neither the sequence nor the structure of the SOLE influences splicing or EJC assembly at the first splice junction in vitro. Furthermore, EJC proteins present in nuclear extracts from Drosophila embryos and Kc cells were deposited on all in vitro-spliced mRNAs tested, irrespective of their sequence; therefore, all the factors required for Drosophila EJC deposition are present in or associated with the nucleus. Notably, a recent study involving genome-wide RNA immunoprecipitation from Drosophila S2 cell extracts suggests that EJC stability may depend on specific RNA sequences found in a subset of Drosophila RNAs that undergo nonsense-mediated decay (Saulière, 2010). Thus, it is possible that the SOLE regulates the association of the EJC with oskar, for example, by recruiting crucial cytoplasmic factors to selectively stabilize the complex on the mRNA in vivo. Conversely, the EJC or spliceosomal factors such as RNA helicases might be required for the SOLE to adopt and stably maintain its secondary structure, which is crucial for RNP motility and posterior localization of the mRNA. Finally, it is possible that a SOLE-binding trans-acting factor cooperates with the EJC for localization (Ghosh, 2012).

Analysis of the motion of both localizing and non-localizing oskar RNPs revealed no detectable difference in the directionality of their motion. It should be noted that, although the numerical values of the average net velocity vectors that were obtained were similar to those previously reported for localizing mRNAs, no statistically significant net posterior vector was detected for any RNPs, with the exception of oskPSLzc. A more detailed analysis will be necessary to resolve this discrepancy. The accumulation of runs within the vicinity of the posterior pole and the posterior-ward translocation of the center of the mass indicate that even the non-localizing mRNAs are displaced toward the posterior pole by a mechanism that remains to be understood (Ghosh, 2012).

Other motion parameters, however, were affected. The travel distance of individual non-localizing oskΔi(1,2,3) and oskPSLz RNPs was substantially shorter and the motile fraction was greatly reduced compared with the localizing oskΔi(2,3) RNPs. Combined, these reductions greatly affect transport efficiency, as has been reported in the case of mutants in the EJC core components (Zimyanin, 2008), and are probably the cause of oskar mRNA mislocalization. Despite the apparent nonzero motility of these mutant RNPs (~17% for oskΔi(1,2,3) and ~29% for oskPSLz), ultimately, a small amount of oskar mRNA accumulated at the posterior pole. A possible explanation for this is that, although the center of mass of the mRNA translocates toward the posterior, the growth of the oocyte along the AP axis is such that the increase in oocyte length exceeds the rate of displacement of the RNPs, suggesting that there is a dynamic threshold crucial for oskar mRNA localization (Ghosh, 2012).

Consistent with the rescuing effect of the compensatory mutation in oskPSLzc on oskar mRNA localization, a restoration was observed of the motion parameters of oskPSLzc*GFP particles to near-wild-type levels and a rate of posterior-ward translocation of the mRNA was similar to that of wild-type particles. Taken together, these observations indicate that the SOLE acts in the localization process by modulating the motility of oskar particles. The changes in transport efficiency of the non-localizing mRNAs are manifest in three different ways: (1) the fraction of motile particles, (2) the duration of the runs and (3) the speed of the runs. These defects might stem from compromised mechanoenzyme activity caused by a lack of associated motors, a failure to maintain the motors' activity or an improper balance of opposite polarity motors. Notably, the reduction observed in the average velocity is due to a relative increase in a slow-moving, short-displacing population of RNPs in oskPSLz and oskΔi(1,2,3) oocytes indicating functional defects in oskar transport. Because KHC is responsible for 70%-80% of the runs, the 60% decrease in the fraction of motile particles in the oskPSLz mutant indicates a severe abrogation of KHC function in oskar transport upon loss of SOLE structural integrity. In contrast, all SOLE mutant RNAs are enriched in the oocyte, indicating that they can associate with the Egl-BicD-dynein complex for their transport from the nurse cells to the oocyte. However, as it was not possible to assess whether their enrichment in the oocyte is quantitatively normal, it remains unclear whether integrity of the SOLE is a requirement of kinesin exclusively or of dynein-based transport of oskar RNPs as well. Analysis of oskar RNP motility with high-temporal resolution imaging may resolve the question of whether the defects observed in the SOLE mutants are of a structural or functional origin and help to elucidate the underlying mechanism(s) (Ghosh, 2012).

Many mRNA localization elements have been predicted to form secondary structures crucial for their function. The observation that SOLE activity depends on the secondary structure of the PS suggests that, as in the case of the K10 transport and localization sequence (TLS) (Bullock, 2010), the structure of the SOLE may be the crucial determinant recognized by the transport machinery. Consistent with this, the bipartite sequence constituting the SOLE stem is conserved across Drosophila species, including at wobble positions. Furthermore, the sequences flanking the first intron of Anopheles, Aedes and Culex oskar homologs, which show little sequence similarity with Drosophila oskar, are predicted to adopt a secondary structure that would be similarly positioned next to the presumed EJC deposition site in those mRNAs, which localize to the posterior pole plasm. To date, oskar is the only mRNA shown to depend on the EJC and a splicing-dependent localization signal for its localization. It will be of interest to look beyond oskar for similar splicing-dependent structures that might cooperate with the EJC in localization of other mRNAs. Alternative splicing has been shown to determine the inclusion of exons containing localization elements in a subset of stardust mRNA isoforms; it could similarly regulate mRNA localization by promoting formation of SOLE-like structures in specific tissues (Ghosh, 2012).

Most localized mRNAs, such as bicoid, gurken and pair-rule transcripts in Drosophila, and ASH1 mRNA in Saccharomyces cerevisiae, require a single type of motor—dynein or myosin, respectively—for localization. In contrast, oskar mRNA, which depends on dynein for its translocation from the nurse cells to the oocyte, switches to KHC within the oocyte for its transport to the posterior pole. This analysis shows that the EJC and SOLE become important for the KHC-dependent step of oskar transport, after dynein-dependent mRNA transport into the oocyte has occurred. Future work should determine whether the SOLE has a structural role in KHC recruitment to oskar RNPs or an important regulatory role in controlling the switch from a dynein to a KHC-dependent mode of transport (Ghosh, 2012).

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain

Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport. This work investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. It is shown that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, it was shown that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, these data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations. It is concluded Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons (Mochizuki, 2011).

Active transport and delivery of molecular and cellular components from the soma to the distinct cytoplasmic compartments is critical not only for synaptic function in mature neurons but also for axonal elongation and guidance in developing neurons. The kinesin-mediated anterograde transport plays a major role in the active traffic in the developing neurons, delivering a wide range of cargos along the axon, including synaptic vesicles, mitochondria, cytoskeletal elements, and mRNAs (Mochizuki, 2011).

This work has demonstrated preferential expression of Unc-51 and kinesin motor proteins in larval MBs, and has shown that loss of unc-51 activity causes severe defects in kinesin-mediated transport in developing MB neurons, while dynein/dynactin-mediated retrograde transport is unaffected in unc-51 mutant MBs. In addition, loss of unc-51 activity results in disintegration of axonal bundles and aberrant extensions of dendrites in both MB and AL neurons. These results suggest that unc-51 is essential for the development of brain neurocircuitries, participating in molecular and/or cellular mechanisms that regulate the formation of the complex structures such as MBs. Indeed, the results demonstrate that unc-51 is essential for the specific intracellular localization of axonal fasciculation and guidance molecules such as Fas II (Mochizuki, 2011).

Although previous studies have demonstrated that unc-51 has diverse functions in developing neurons, this analyses of the Klc+/- unc-51+/- double heterozygotes clearly demonstrate that suppression of kinesin-mediated transport results in dendritic overextensions. Concomitant suppression of unc-51 and Klc also causes axonal transport defects that are reminiscent of unc-51 mutants. Furthermore, the double heterozygotes exhibit mislocalization of Fas II in both the calyx and the proximal peduncle. This recapitulates the salient phenotype of unc-51-/- mutants, and argues that defective kinesin-mediated transport is the major molecular process that underlies the developmental defects in the unc-51 mutants. In contrast, Klc+/- unc-51+/- double heterozygotes failed to exhibit a full range of the unc-51 mutant phenotypes. Although the possibility cannot be excluded that other molecular processes are involved in the unc-51 mutant phenotypes, the weaker phenotypes of the double heterozygotes might be accounted by partial suppression of the gene activity given that wild-type alleles are retained at a half dosage for both genes. Furthermore, the result that Klc null mutant clones exhibited dendritic and axonal defects that were reminiscent of the unc-51 mutant clones clearly confirms the importance of the kinesin-mediated transport in brain development. It is also noteworthy that, as with Klc mutant clones, Khc null MB clones exhibit similar yet more pronounced defects, although the critical requirement of the Khc activity for neuroblast division hampers a precise assessment of Khc function in axodendritic development. These results as a whole highlight the importance of kinesin-mediated vesicle transport in the development and wiring of complex networks in the brain (Mochizuki, 2011).

Previous studies have demonstrated that unc-51 plays an essential role in axonal transport by mediating the assembly of the cargos and the kinesin motor proteins. In unc-51 mutants, membrane vesicle transport is severely affected, resulting in accumulation of synaptic vesicles in the larval segmental nerves. Loss of unc-51 activity also causes aggregation of Rab5-positive membranes in the segmental nerves. Notably, as in the mutant MBs, kinesin motors are accumulated in the mutant segmental nerves while overall mitochondrial localization was unaffected. Genetic studies have shown that the wild-type but not a kinase-deficient form of the unc-51 transgene rescues the transport defect in synaptic vesicles in the mutant segmental nerves. Moreover, the kinase activity of Unc-51 is critical for phosphorylation of an adaptor molecule, Unc-76/FEZ, which mediates the assembly between synaptic vesicle cargos and the kinesin motor complex. The finding that the dendritic and axonal defects in MB neurons are rescued by the wild-type but not by the kinase-deficient unc-51 transgene suggests that a similar phosphorylation-dependent regulatory mechanism is involved in the intracellular transport in developing MB neurons. In line with this, it is noteworthy that Unc-76 is preferentially expressed in developing MB neurons, in which it colocalizes with Unc-51 and kinesin motor proteins in the core fibers. Intriguingly, both Unc-51 and Unc-76 are downregulated in the mature neurons that surround the core fibers as they mature and shift to the peripheral layers. Concomitantly, both Khc and Klc are downregulated in the mature MB neurons, suggesting dynamic control of the expression of the molecular components that mediate active vesicle transport in developing MB neurons (Mochizuki, 2011).

Recent genetic studies on the dendritic development of Drosophila larval sensory neurons showed that the microtubule motor protein dynein controls dendritic branching by directing polarized intracellular vesicle trafficking. Dynein is also necessary for the dendrite-specific localization of Golgi outposts, which secretory pathway plays critical roles in dendritic growth and branching. These studies also showed that Rab5 was essential to normal dendritic branching, via its role in controlling endosomal trafficking. The current results show that mutations of unc-51 leads to aberrant accumulations of Rab5-containing endosomes and Golgi components in developing larval MBs. In contrast, polarized dendritic transport of Dscam is not altered in the unc-51 mutant MBs, implying that retrograde dynein/dynactin-mediated transport remains intact in the mutant MB neurons. Moreover, another dendritic molecule, Nod, is correctly targeted to the calyx, clearly indicating that unc-51 is not required for polarized retrograde transport mediated by dynein/dynactin in developing MB neurons. These results are consistent with previous observations that unc-51 fails to interact with retrograde motor genes such as dynein heavy chain and Lissencephaly-1 in segmental nerves (Mochizuki, 2011).

In contrast, previous studies found that khc mutants showed dendritic branch abnormalities that were almost identical to those of dynein light intermediate chain (Dlic) mutants, with reduced arbors and a marked shift in branching activity in the proximal area within the arbors. In the khc mutants, the dynein complexes become aggregated distally, suggesting that kinesin plays a role in recycling dynein proximally after it has carried its organelle cargo distally. These phenotypes seem to contrast with those observed in unc-51 mutant MB neurons, which show dendritic overextensions with normal dynein/dynactin-mediated transport. The exact mechanisms underlying these discrepancies are unknown, but this might suggest different regulatory processes for kinesin-mediated transport that operate in the distinct cellular contexts of the peripheral sensory neurons and MB neurons. It was showm that unc-51 mutation resulted in varying degrees of axonal membrane defects that were dependent on the cargo. It is possible that Unc-51 differentially regulates the transport of specific cargo subsets by phosphorylation of distinct groups of adaptor proteins in different cell types (Mochizuki, 2011).

Studies in Drosophila identified unc-51 as a homolog of the yeast atg1, and suggested that Unc-51 kinase activity is required for the induction of autophagy. It has been shown that autophagy positively regulates synapse development at the Drosophila neuromuscular junction. Mutations in autophagy genes including atg1/unc-51, caused significant reduction in terminal branching of the segmental motoneurons, with reduced numbers of boutons. In contrast, this study has shown that single-cell analysis of AL-PNs shows that loss of unc-51 activity results in an increase in the number of the calyx branches. The results also demonstrate that the distribution of an autophagy marker is not altered between the wild-type and the unc-51 mutant MBs. These results argue against autophagy as an underlying mechanism of the axodendritic abnormalities in the unc-51 mutant larval brain, and are consistent with a previous report that autophagy is not involved in Unc-51-mediated regulation of axonal transport (Mochizuki, 2011).

In C. elegans, mutations in unc-51 cause diverse axonal defects, including premature termination, abnormal trajectories, and extra axon branches. Developing neurites accumulate abnormal vesicles and cisternae, suggesting underlying defects in membrane vesicle trafficking. Intriguingly, Unc-51 directly interacts with Unc-14, a RUN domain protein, to regulate axonal elongation and guidance, and mutations in unc-14 cause neurite growth and guidance defects that are very similar to those of unc-51. Unc-14 regulates vesicle transport and localization by binding to Unc-16/JIP3/JSAP1, which is a cargo adaptor for the kinesin motor proteins. Recent studies have shown that RUN domain proteins function as effectors of Rap and Rab GTPases in the control of membrane trafficking, highlighting the importance of vesicle trafficking in the regulation of axonal growth and guidance. Several studies have suggested that Unc-51 plays an essential role in the delivery of specific receptors for axonal guidance molecules. Together with Unc-14, Unc-51 regulates the subcellular localization of the Netrin receptor/Unc-5 in C. elegans. Thus, in unc-14 and unc-51 mutants, the Netrin receptor accumulates in neural cell bodies rather than at axonal termini, causing severe guidance defects in the DD/DV neuron. Unc-51 also interacts with the kinesin-related Vab-8 protein, which regulates anterior-posterior migration of C. elegans mechanosensory neurons through the regulation of another Netrin receptor Unc-40/Dcc and the Slit receptor Sax-3/Robo. Vab-8 controls cell-surface expression of Sax-3/Robo in the growth cones of touch neurons through interaction with Unc-73/Trio, a guanine nucleotide exchange factor for Rac. Consequently, peptide-mediated interference with the Vab-8 and Unc-51 interaction in worm neurons blocked axonal outgrowth and posteriorly directed guidance (Mochizuki, 2011).

In mouse, Unc51.1/Ulk1 is expressed in granule cells in the cerebellar cortex, and retroviral infection of immature granule cells with a dominant-negative Unc51.1 caused inhibition of neurite outgrowth both in vitro and in vivo. Subsequent molecular studies showed that Unc51.1 binds to SynGAP and Syntenin, the latter of which, in turn, binds Rab5 GTPase to tether the Unc51.1/SynGAP/Rab5 complex to the vesicular membrane. Immunoelectron microscopy of granule cells provided evidence that Unc51.1 indeed associates with membrane vesicles. Moreover, SynGAP stimulates the GTPase activity of Rab5, and overexpression of SynGAP in cultured cerebellar granule neurons leads to truncated neurites and disorganized vesicular compartments. This suggests that the Unc51.1-containing protein complex governs axonal elongation and pathfinding by modulating the Ras-like GTPase signaling pathway and the Rab5-mediated endocytic pathway in developing neurons (Mochizuki, 2011).

The importance of Unc-51 in the regulation of vesicle trafficking is further supported by the finding that suppression of Unc-51 activity leads to increased neurite branch formation and shortened axons in cultured mouse dorsal root ganglia neurons. Both Unc51.1 and Unc51.2 are localized to vesicular structures in growth cones in sensory axons, in which Unc51.1 promotes endocytosis of the neurotrophic tyrosine kinase receptor Ntrk1/TrkA through a non-clathrin mediated pathway, presumably through the interaction of Unc51.1 with SynGAP and Rab5. Moreover, Unc51.1 also interacts with the Golgi-associated ATPase enhancer of 16 kD (Gabarapl2/Gate-16), which is an essential factor for intra-Golgi transport\. Unc51.1 also interacts with the gamma-2 subunit of the GABA-A receptor associated protein (GABARAP), which is again involved in the regulation of receptor trafficking. Together with the current findings in the Drosophila brain, these studies highlight the functional importance of the Unc-51 family proteins in the endocytic processes that regulate diverse signaling events during axonal elongation, fasciculation, and guidance. Loss of the Unc-51 activity is likely to perturb the trafficking of multiple types of post-Golgi vesicles and lead to severe disruption of the controlled delivery of essential axonal growth/guidance receptors to the different compartments of growing neurons. Elucidation of the exact molecular components that are involved in Unc-51-mediated regulation of vesicle transport is an important subject for future studies. It is envisaged that studies in Drosophila will continue to provide critical insights into the conserved molecular mechanisms of coordinate regulation of membrane vesicle trafficking and axon growth/guidance in developing neurons (Mochizuki, 2011).

Disruption of axonal transport perturbs Bone Morphogenetic Protein (BMP) - signaling and contributes to synaptic abnormalities in rwo neurodegenerative diseases

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. This study shows that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). Components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility is also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also show synaptic morphological defects. Strikingly TKV motility and p-Mad signaling are disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP; see Drosophila Appl) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins show accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together these results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exists. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases (Kang, 2014 - Open access: 25127478).

Effects of Mutation or Deletion

The in vivo function of the microtubule motor protein kinesin was examined in Drosophila using genetics and immunolocalization. Kinesin heavy chain mutations cause abnormal behavior and lethality. Mutant larvae exhibit loss of mobility and tactile responsiveness in the most posterior segments, followed by general paralysis and death during larval or pupal development. Adults homozygous for a temperature-sensitive allele also exhibit a loss in mobility and sensory responses. The data indicate that kinesin function is essential and suggest that kinesin has an important role in the neuromuscular system, perhaps as a motor for axonal transport. The possibility of more general cellular functions remains open, but observation of embryogenesis and morphogenesis in Khc mutants suggests that mitosis and the cell cycle can proceed in spite of impaired kinesin function. Immunolocalization suggests that kinesin may have some general cellular functions but that it is not a major component of mitotic spindles (Saxton, 1991).

Kinesin is believed to generate force for the movement of organelles in anterograde axonal transport. The identification of genes that encode kinesin-like proteins suggests that other motors may provide anterograde force instead of or in addition to kinesin. To gain insight into the specific functions of kinesin, a study was made of the effects of mutations in the Kinesin heavy chain gene on the physiology and ultrastructure of Drosophila larval neurons. Mutations in Khc impair both action potential propagation in axons and neurotransmitter release at nerve terminals but have no apparent effect on the concentration of synaptic vesicles in nerve terminal cytoplasm. Thus kinesin is required in vivo for normal neuronal function and may be active in the transport of ion channels and components of the synaptic release machinery to their appropriate cellular locations. Kinesin appears not to be required for the anterograde transport of synaptic vesicles or their components (Gho, 1992).

To study the relationship between conventional kinesin's structure and function, 13 lethal mutations were identified in the Drosophila kinesin heavy chain motor domain and a subset was tested for effects on mechanochemistry. S246F is a moderate mutation that occurs in loop 11 between the ATP- and microtubule-binding sites. While ATP and microtubule binding appear normal, there is a 3-fold decrease in the rate of ATP turnover. This is consistent with the hypothesis that loop 11 provides a structural link that is important for the activation of ATP turnover by microtubule binding. T291M is a severe mutation that occurs in alpha-helix 5 near the center of the microtubule-binding surface. It impairs the microtubule-kinesin interaction and directly effects the ATP-binding pocket, allowing an increase in ATP turnover in the absence of microtubules. The T291M mutation may mimic the structure of a microtubule-bound, partially activated state. E164K is a moderate mutation that occurs at the beta-sheet 5a/loop 8b junction, remote from the ATP pocket. Surprisingly, it causes both tighter ATP-binding and a 2-fold decrease in ATP turnover. It is proposed that E164 forms an ionic bridge with alpha-helix 5 and it is speculated that this region helps coordinate the alternating site catalysis of dimerized kinesin heavy chain motor domains (Brendza, 1999).

To probe the mechanochemical mechanisms employed by kinesin random mutagenesis of Drosophila and DNA sequencing were used to identify amino acid changes in Khc that impair motor domain function in vivo. Of the 40 recessive lethal mutations studied, 10 have missense amino acid changes in the motor domain. Four of those were selected for further characterization. The four mutations range from a severe allele that causes a near complete loss of function to a mild allele that causes only a partial loss of function. After expression and purification of the motor domains fused to a biotin motility anchor, microtubule gliding assays showed that the mutant motors retained some mechanochemical functions. However, the relative rates of gliding did not agree well with the relative severities of the in vivo phenotypes. It is suspected that gliding rates are extremely sensitive to the presence of inactive motors that may interfere with microtubule gliding as well as the orientation of the active motors on the slide. Because of the combined effects of these two phenomena, the rates measured may not reflect accurately the mechanochemistry of the active majority. Using a different motor domain construct without the biotin anchor and optimized for purification of active proteins, steady-state kinetic analysis of the mutant proteins has revealed defects whose relative severities generally correlated with the relative severities of the in vivo phenotypes. These results suggest that the changes in steady-state kinetics of ATP turnover that were measured in vitro are physiologically relevant (Brendza, 1999).

To interpret the kinetic effects of the mutations, the evolutionary conservation of the amino acids that are changed and their positions in the atomic structure of a motor domain dimer were all considered. The allele that was mildest in vivo, Khc37, has an aspartic acid to asparagine change at the junction of alpha4 and L12. Asparagine is less polar than aspartic acid but their side chains are very similar in size. The steady-state kinetic analysis showed relatively mild effects, however, a change of the corresponding human Khc residue to alanine (nonpolar and small) caused a 2-fold reduction in K0.5, MT. Combined, these results indicate that both the polarity and the size of this residue are important for correct microtubule-Khc interaction. Furthermore, since Khc37 clearly causes axonal transport defects and semilethality, these results suggest that even slight changes in kinetics can have significant consequences in vivo (Brendza, 1999).

The next mildest allele, Khc17, changes a serine to a phenylalanine in L11. Because L11 has not been resolved by crystallography, the position and orientation of the serine side chain is difficult to predict. However, one can assume that the change disrupts any interactions that the serine normally has because the phenylalanine side chain is dramatically larger and more hydrophobic. The S246F mutation causes a substantial decrease in kcat (5.9 versus 20 s-1 for wild-type), kcat/Km,ATP (0.065 versus 0.2 µM-1 s-1), and kcat/K0.5,MT (3.6 versus 25 µM-1 s-1), yet both the Km,ATP and K0.5,MT are close to wild-type. These results suggest that Khc17 binds ATP and the microtubule lattice relatively normally, yet a key step for ATP turnover is defective. This interpretation is consistent with the hypothesis that L11 acts as a structural link to couple microtubule binding to activation of the hydrolytic cycle (Brendza, 1999).

The most severe motor domain allele, Khc4, changes a threonine in alpha5 to a methionine (T291M). The methionine side chain is larger than that of threonine and more hydrophobic. Furthermore, the threonine side chain should form a hydrogen bond, perhaps with an amino acid in alpha4, and the methionine side chain does not hydrogen bond. Thus, the T291M mutation probably changes the orientations of alpha5, alpha4, and L12; this in turn cause shifts in other structural elements. The kinetic changes caused by the mutation suggest that the structures of both the ATP-binding pocket and the microtubule-binding site are altered to mimic a partially activated site. Consistent with this interpretation is the observation that there is a substantial increase in the rate of steady-state ATP turnover in the absence of microtubules. These results and the fact that Thr291 is invariant in the kinesin superfamily suggest that it plays a key role in the transmission of structural changes between the microtubule- and ATP-binding sites (Brendza, 1999).

The Khc23 allele, less severe than Khc4 and more severe than Khc17, changes a negatively charged glutamic acid to a positively charged lysine (E164K) at the ß5a/L8b junction. The glutamic acid side chain is solvent exposed and remote from the ATP-binding pocket, yet the change to lysine causes tighter ATP binding and a 2-fold reduction in ATP turnover rate. A change of the corresponding glutamic acid in human KHC to an uncharged alanine also causes a 2-fold reduction in ATP turnover rate. Thus, it may be the loss of the negative charge in Khc23 rather than the gain of the positive charge that causes the slow ATP turnover. Combined, these observations suggest that Glu164 normally participates in an ionic interaction that influences the structure of the nucleotide-binding pocket during the ATPase cycle (Brendza, 1999).

An examination of Khc crystal structures reveals a possible mechanism for the influence of Glu164 on nucleotide binding. The elongated, negative Glu164 side chain extends away from ß5a/L8b and the dimer interface toward the center of the microtubule-binding surface. It comes into close proximity with the elongated, positive side chain of Arg292, which projects from helix alpha5 toward ß5a/L8b. Given their proximity and opposite charges, Glu164 and Arg292 could interact to form an ionic link between alpha5 and ß5a/L8b. It is worth noting that in human KHC, replacement of the Arg292 equivalent by an uncharged alanine (R284A), like the E164K change, causes a 2-fold reduction in ATP-turnover rate. It is also noteworthy that this Glu164/Arg292 amino acid pair is highly conserved in the Khc and several other NH2-terminal kinesin subfamilies, but is very divergent in the COOH-terminal kinesin subfamily. It is proposed that a Glu164-Arg292 ionic bridge coordinates the positions of L8 and alpha5, and that the effect of the E164K mutation on ATP binding is due to a misorientation of alpha5, which as discussed above for T291M, can influence the structure of the nucleotide-binding pocket (Brendza, 1999).

It is interesting to consider the possibility that the ß5a/L8b to alpha5 bridge is one part of an extended chain that links the nucleotide-binding pockets of dimerized KHCs. There is a possibility of an ionic interaction between positive residues in L8b of one motor domain and negative residues in L10 of the partner motor domain. This linkage would be transient because it would need to break during the mechanochemical cycle to allow simultaneous binding of both motor domains to the microtubule and then would presumably reform in the opposite orientation. Strand 7 of the central ß-sheet links L10 directly to the switch II element of the nucleotide-binding pocket. Thus, a linkage extending from the switch II region of one head through L10 to L8b and alpha5 of the other head could be important in coordinating the activities of the two nucleotide-binding pockets. A lack of sequence conservation in L10 of KHCs from different species casts some doubt on this L8b-L10 linkage. However, the linkage of L8b to some part of the partner motor domain remains an attractive possibility for coupling dimer interactions to nucleotide and perhaps microtubule binding through shifts in alpha5. Steady-state kinetic analysis does not provide the temporal resolution required to identify specific steps in the ATPase cycle that are altered by the mutation (Brendza, 1999 and references therein).

In summary, 9 amino acid residues have been identified that are critical for kinesin function in Drosophila. The results of kinetic analysis of lethal amino acid substitutions at some of those sites have revealed that the activity of the nucleotide-binding pocket is altered by structural changes on the side of Khc that binds microtubules. The results also support the idea that structural elements distant from one another within a motor domain and between dimerized motor domains are tightly integrated such that defects in ATP turnover affect microtubule binding and defects in microtubule binding affect ATP turnover. The tight coupling of these activities is not surprising when one considers the demands placed on kinesin in vivo. Small organelles can use only a few kinesin molecules to sustain processive transport, and missteps are costly, as illustrated by the effects of even mild kinesin mutations on axonal transport. It appears that a few stalled axonal organelles can trigger massive traffic jams in Drosophila axons that cause serious declines in neuron function. Mechanistic studies of mutated Khcs are in progress to evaluate further the kinetic-structural relationships that underlie the precise coordination required for processive movement (Brendza, 1999).

To analyze the effects of mutation of Drosophila Kinesin heavy chain, all assays were performed using two independently isolated null alleles of the Khc. Both produced the same results, and all defects could be rescued by a transgenic copy of wild-type Khc (P{mini-w+, Khc+}). The Khc27 allele, which is recessive lethal, has a nonsense mutation at codon 65 that presumably halts translation and prevents KHC synthesis (Brendza, 1999). The molecular lesion in Khc20, another recessive lethal allele, has not been identified, but it causes a complete loss of function by genetic criteria and produces phenotypes identical to those of Khc27 in all assays to date. Null alleles of Khc should exert the same phenotypic effects as a deletion of the Khc locus (Saxton, 1991). Using the time course of lethality to assess levels of KHC function in homozygous or hemizygous animals, Khc20 and Khc27 are equivalent to a deletion [Df(2R)Jp6]. A more sensitive method to determine whether Khc20 and Khc27 behave like a Khc deletion is to compare the effects of heteroallelic combinations with a hypomorphic Khc allele (e.g., Khc6). The lethal phase profiles for populations of larvae that are Khc6/Khc20, Khc6/Khc27 or Khc6/Df(2R)Jp6 are indistinguishable. This confirms that the Khc20 and Khc27 alleles are functionally null, equivalent to a deletion of the Khc gene (R. P. Brendza, 2000a).

All Drosophila cells tested to date express KHC (Saxton, 1988). Tests of eye and wing discs by immunofluorescence indicate that KHC is distributed throughout the cytoplasm and is excluded from the nucleus as has been demonstrated for embryonic cells (Saxton, 1991). Immunoelectron microscopy suggests an even distribution throughout the cytoplasm of adult photoreceptor cells and exclusion from rhabdomeres (S. Benzer, personal communication to R. P. Brendza, 2000a). To study the effects of a loss of KHC on the proliferation of various cell types, an FLP recombinase, site-specific mitotic recombination system with Khc20 and Khc27 (referred to interchangeably below as Khcnull) was used to generate single Khcnull/Khcnull cells in Khcnull/Khc+ larvae. The proliferative capacities of single Khcnull/Khcnull cells were assessed by comparing the amount of adult tissue each could generate to the amount generated by equivalent control cells. The cells studied in detail included those in the eye imaginal disc, the wing imaginal disc, and the abdominal histoblast nests that form abdominal bristles (R. P. Brendza, 2000a).

To determine whether Khcnull cells in the developing eye can proliferate normally, mitotic recombination was used to generate pairs of sister cells, one homozygous null and the other homozygous wild type for Khc. The induction of mitotic recombination (1-2 d after egg laying) leads to adult progeny (10 d after egg laying) with Khcnull eye clones of substantial size. Nevertheless, after the loss of Khc gene function, at least 11 rounds of cell growth and division can be completed at normal rates. This suggests that kinesin is not important for the growth or division of eye imaginal cells. Khcnull clones were also studied in the portion of the wing imaginal disc that gives rise to the anterior wing margin. To mark wing clones, a wild-type yellow transgene was linked to the Khc+ allele in a mutant yellow background. Consequently, bristles in the null clones were yellow, whereas bristles in sister clones and in nonrecombinant tissue were black. Again, Khcnull cells in the wing imaginal disc can proliferate normally (R. P. Brendza, 2000a).

To address the possibility that dividing imaginal cells might not challenge the secretion pathway sufficiently to reveal kinesin's functions, post-mitotic differentiating cells that depend heavily on membrane growth and secretion were analyzed. The Drosophila compound eye consists of approximately 750 ommatidia, each composed of a columnar cluster of eight elongated photoreceptor cells surrounded by a thin layer of pigment cells. Each photoreceptor cell has a light-sensing rhabdomere, which is a tightly packed array of 60,000 microvilli that extends along the length of the ommatidial column (~50-100 µm). The specification of the various cells of an ommatidium and their differentiation require precise cell-cell signaling and a massive expansion of plasma membrane; from the ~150 µm2 of an imaginal cell to ~10,000 µm2 for a mature photoreceptor. Based on these considerations, even moderate defects in the secretory pathway should have dramatic consequences during eye differentiation (R. P. Brendza, 2000a and references therein).

To look for signs of defects in the adult eye caused by a loss of kinesin function, mitotic clones were examined by electron microscopy. Matings were arranged to produce two types of sibling progeny that carried Khcnull clones: a control class with a rescuing Khc+ transgene and a test class without the transgene. Test and control clones were mapped and photographed with a light microscope and then examined with a scanning electron microscope. Slightly roughened eye surfaces were seen within test clones, suggesting defects in the underlying cells. To characterize those defects, 25 test and 5 control clones from newly eclosed flies were sectioned and examined by TEM. No defects were detected in control clones. In test clones, ~20% of the ommatidia were missing one or two photoreceptors. This loss, which is characteristic of mild defects in postmitotic differentiation, reduced the total number of photoreceptor cells in the clones by 5%. In flies aged for >2 wk after eclosion, degenerating photoreceptors were seen at a low frequency. This age-dependent degeneration may be a result of defective fast transport in photoreceptor axons. In addition, some photoreceptors in null clones showed structural defects, including disordered packing of microvilli and split or buckled rhabdomeres. The number of photoreceptors with such abnormal rhabdomeres varied from clone to clone but never exceeded 5%-10%. The missing and malformed photoreceptors in newly eclosed flies altered the shapes of their ommatidia and hence caused disorder in the ommatidial array, which accounted for some of the surface roughness seen by SEM. These defects appeared equally severe in small and large clones, confirming that the decline of KHC function in a null clone was fairly complete after only a few cell cycles (R. P. Brendza, 2000a).

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr291 of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr291 results in complete loss of function in vivo. In vitro, the T291M mutation, Khc4, disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, 1999). The pre-steady-state kinetic analysis presented in this study shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin (Brendza, 2000).

A truncated domain of the alpha-subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH392) extends to amino acid 392 and contains the complete N-terminal region of kinesin which is highly conserved between species. The DKH392 construct includes an additional 52 amino acids beyond the minimal motor domain of 340 amino acid residues that has been previously characterized as DKH340. DKH340 is a monomer in solution, but DKH392 is a dimer. In the presence of adenosine 5-(beta,gamma-imido)triphosphate, DKH392 binds to microtubules with a stoichiometry of two head domains (one DKH392 dimer) per tubulin heterodimer in contrast to the tight binding of one DKH340 per tubulin heterodimer. Electron microscopy indicates that both DKH340 monomers and DKH392 dimers decorate microtubules with a periodicity of 8 nm (Huang, 1994b).

A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)triphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. A model is presented in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP, and thus it is likely to be in a conformational state that is not produced during steady state ATP hydrolysis (Huang, 1994a).

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in these diseases (Hurd, 1996b).

To investigate the possibility that kinesin transports vesicles bearing proteins essential for ion channel activity, the effects of kinesin (Khc) and ion channel mutations were compared in Drosophila using established tests. Khc mutations produce defects and genetic interactions characteristic of paralytic (para) and maleless (mle) mutations that cause reduced expression of function of the alpha-subunit of voltage-gated sodium channels. Like para and mle mutations, Khc mutations cause temperature-sensitive (TS) paralysis. When combined with para or mle mutations, Khe mutations cause synthetic lethality and a synergistic enhancement of TS-paralysis. Furthermore, Khc mutations suppress Shaker and ether-a-go-go mutations that disrupt potassium channel activity. In light of previous physiological tests that show that Khc mutations inhibit compound action potential propagation in segmental nerves, these data indicate that kinesin activity is required for normal inward sodium currents during neuronal action potentials. Tests for phenotypic similarities and genetic interactions between kinesin and sodium/potassium ATPse mutations suggest that impaired kinesin function does not affect the driving force on sodium ions. It is hypothesized that a loss of kinesin function inhibits the anterograde axonal transport of vesicles bearing sodium channels (Hurd, 1996a).

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein (see Dynein heavy chain 64C) is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. The functions of the cytoplasmic dynein heavy chain (cDhc64C) and the Glued component of the dynactin complex has been tested with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport (Martin, 1998).

To establish the major body axes, late Drosophila oocytes localize determinants to discrete cortical positions: bicoid mRNA to the anterior cortex, oskar mRNA to the posterior cortex, and gurken mRNA to the margin of the anterior cortex adjacent to the oocyte nucleus (the 'anterodorsal corner'). These localizations depend on microtubules that are thought to be organized such that plus end-directed motors can move cargoes, like oskar mRNA, away from the anterior/lateral surfaces and hence toward the posterior pole. Likewise, minus end-directed motors may move cargoes toward anterior destinations. Contradicting this, cytoplasmic Dynein, a minus-end motor, accumulates at the posterior. Disruption of the plus-end motor kinesin I causes a shift of dynein from posterior to anterior. This provides an explanation for the dynein paradox, suggesting that dynein is moved as a cargo toward the posterior pole by kinesin-generated forces. However, other results present a new transport polarity puzzle. Disruption of kinesin I causes partial defects in anterior positioning of the nucleus and severe defects in anterodorsal localization of gurken mRNA. Kinesin may generate anterodorsal forces directly, despite the apparent preponderance of minus ends at the anterior cortex. Alternatively, kinesin I may facilitate cytoplasmic dynein-based anterodorsal forces by repositioning dynein toward microtubule plus ends (Brendza, 2002).

To better understand microtubule-based localization processes in Drosophila oocytes, the localization of kinesin I was studied with an antiserum that binds its motor subunit, kinesin heavy chain (Khc). An even distribution of Khc is seen throughout the germline cells of the germarium and early egg chambers. Staining was usually more intense in the somatic follicle cells that enclose the egg chambers and was particularly strong in polar follicle cells. Beginning in stage 8 and continuing through stage 10A, Khc is most concentrated at the posterior pole of the oocyte. A small concentration also appears in the anterodorsal corner. Disruption of Khc expression in clones of cells by mitotic recombination with a null allele of the Khc gene showed that the posterior Khc is a product of the germline and not of the posterior follicle cells (Brendza, 2002).

Previous studies of microtubules in late-stage oocytes suggest that microtubule minus ends are most concentrated at the anterior and least concentrated at the posterior pole. In addition, tests of the localization of ß-galactosidase fused to the motor domains of Khc or Nod suggest that plus-end transport is directed toward the posterior pole and minus-end transport is directed toward the anterior margin. This is consistent with posterior accumulation of Khc and with the disruption of posterior oskar mRNA localization reported in Khc mutants. However, in apparent contradiction, cytoplasmic dynein, which is minus end-directed, has also been shown to accumulate at the posterior pole in late-stage oocytes (Brendza, 2002).

To test the possibility that dynein is carried toward the posterior pole by kinesin I, the distribution of cytoplasmic dynein heavy chain (cDhc) and Khc was compared in late-stage Khc mutant oocytes, produced by Khc null germline clones. In the Khc mutants, cDhc staining shows little or no posterior localization; rather, it accumulates strongly at the anterior. Anti-tubulin staining indicates that the anterior-posterior gradient of microtubules is not disrupted in Khc null oocytes. Therefore, the shift of dynein to the anterior in Khc mutants suggests that kinesin I is responsible for moving cytoplasmic dynein away from minus ends at the anterior and thus moving it toward the posterior pole (Brendza, 2002).

Examination of the chorions of eggs produced by Khc null germline clones has suggested defects in dorsal-ventral axis formation. Proper dorsal pole specification within the oocyte induces follicle cells to differentiate into a pair of dorsal respiratory appendages near the anterior end of mature eggs. Of 359 eggs from Khc null germline clones, only 1% had normal appendages. Of the remainder, 17% had fused appendages, 26% had a rudimentary dorsal bump, and 56% showed no dorsal material. These phenotypes are completely rescued by a wild-type Khc transgene. These results indicate that germline kinesin I has an important role in dorsal pole specification (Brendza, 2002).

Early steps in dorsal specification occur during stage 7. The posterior microtubule-organizing center (MTOC) disassembles, and the oocyte cortex takes on MTOC activity. Microtubules become particularly abundant at the anterior and anterior margins and are least abundant at the posterior. This suggests an anterior-posterior gradient of cortical microtubule minus ends. The nucleus then shifts from the posterior pole to the anterior margin in a microtubule-dependent manner, and gurken mRNA becomes concentrated around the entire anterior margin. Subsequently, during stages 8–10, gurken disappears from most of the anterior margin and becomes concentrated between the nuclear envelope and the adjacent anterior-lateral cortex (the anterodorsal corner) in a microtubule-dependent manner. Gurken protein is expressed and secreted there, inducing dorsal fates in neighboring follicle cells (Brendza, 2002).

In Khc null stage-8 to -10 oocytes, anti-Gurken immunostaining reveals that anterodorsal accumulation is either weak or absent. Consistent with poor Gurken expression, kekkonI mRNA, which is normally induced in anterodorsal follicle cells by Gurken signaling from the oocyte, is weak or absent. These results indicate that Khc in the germline is required for normal anterodorsal Gurken expression and signaling (Brendza, 2002).

The processes underlying anterodorsal Gurken expression were examined by in situ hybridization and light microscopy. During stages 6–8, gurken mRNA shows a normal transition from localization at the posterior to localization at the anterior margin. The anterior signal in stage 8 appears as a ring in both mutants and controls. However, in stage-9 and -10 mutant oocytes, rather than localizing to the anterodorsal corner, the gurken signal is almost always spread evenly across the anterior in a broad diffuse band that has no ring-like profile. This indicates that kinesin I is critical for normal anterodorsal localization of gurken mRNA. Poor expression of Gurken from the mislocalized mRNA, and the consequent lack of dorsalization, is likely to reflect position-dependent translational repression (Brendza, 2002).

The position of the oocyte nucleus on the anterior margin defines the site of gurken mRNA localization and thus is a critical part of the localization mechanism. Nuclear positioning was defective in about 50% of stage-9 and -10 Khc null oocytes. Nuclei appear to accomplish the initial posterior to anterior shift during stage 7; however, a rigorous assessment of nuclear position is difficult in stage 7 because of the small size of the oocyte. To gain further insight, nuclear positioning was compared in wild-type and Khc null stage-8 to -10 oocytes. Although some nuclei were mispositioned in stage-8 mutants, there was a marked shift away from the anterior margin in stages 9 and 10. While these data do not establish whether or not Khc has a minor role in initial anterior migration, the decline in normal positioning during stages 8–10 suggests that kinesin I does help keep the nucleus at the anterior. The poor retention in Khc mutants may reflect defects in the anchoring of the nucleus to the cortex of the anterior margin. It could also reflect a decline in ongoing anterodorsal forces on the nucleus that may be needed to maintain its normal position. Thus, the mechanism of anterodorsal gurken localization requires proper nuclear positioning, microtubules, and kinesin I (Brendza, 2002).

In summary, the results provide several insights into localization processes during mid-late oogenesis: (1) kinesin I colocalizes at the posterior pole with cytoplasmic dynein; (2) kinesin I is required for the posterior localization of cytoplasmic dynein; (3) kinesin I is required for the dorsal localization of gurken mRNA, and (4) kinesin I contributes to the proper anterior positioning of the oocyte nucleus. A role for kinesin in moving dynein toward the posterior pole provides a solution to the paradox of the accumulation of a minus-end motor in an area thought to be a destination for plus end-directed transport. However, a role for kinesin in anterodorsal localization is surprising because of evidence that minus ends are most concentrated there. In particular, a Nod:ß-galactosidase fusion protein that is targeted to microtubule minus ends accumulates around the nucleus and at the anterior margin during stages 8–10. How might a plus end-directed motor participate in localization toward an area dominated by microtubule minus ends (Brendza, 2002)?

Previous reports and recent results suggest that dorsal pole specification requires the minus end-directed motor, cytoplasmic dynein. Hypomorphic mutations that impair the function of Drosophila Lis1, which is known to be required in various systems for dynein/dynactin function in nuclear migration and other motility processes, can cause ventralization of chorions, mislocalization of the nucleus, and failure of anterodorsal gurken localization. Conditional overexpression of a protein that disrupts the dynein/dynactin complex has been shown to cause equivalent, though more severe, defects in those same dorsal specification processes. The fact that the same dorsal pathway phenotypes are caused by germline Khc disruption suggests that kinesin I and cytoplasmic dynein both are required for nuclear positioning and anterodorsal gurken mRNA localization (Brendza, 2002).

The following model is proposed to explain these results. Dynein, which is synthesized in nurse cells, walks along microtubules from nurse cells through connecting ring canals toward microtubule minus ends at the oocyte posterior until stage 4. After the microtubule cytoskeleton reorganizes during stage 7, concentrating minus ends at the anterior cortex, dynein-generated movements are redirected away from the posterior. This drives the nucleus and gurken mRNA to the anterior margin. Materials like dynein and determinant mRNAs, moved by unknown forces, continue to enter the oocyte from nurse cells through the anterior ring canals. Those that need to be distributed toward the posterior and are too large to diffuse efficiently are moved by kinesin I, either directly or by means of cytoplasmic flows. As the oocyte enlarges during late stages, diffusion of the large cytoplasmic dynein/dynactin complex away from anterior minus ends becomes limiting. Thus, active transport of dynein away from the anterior by kinesin or by kinesin-generated cytoplasmic flows becomes critical. In stage-9 and -10 Khc mutant oocytes, dynein is trapped near minus ends at the anterior cortex. Anterior-directed dynein-based forces that act on gurken mRNA, the nucleus, and/or nuclear anchors are reduced, disrupting their normal positioning mechanisms (Brendza, 2002).

If this dynein recycling model is correct, why does a loss of Khc influence nuclear position and disrupt anterodorsal gurken localization but not other putative dynein functions, such as the anterior localization of bicoid mRNA? As with the initial localization of gurken mRNA, dynein-based forces toward the anterior margin may not be sensitive to poor recycling while the oocyte is small. Subsequent anterior localization of bicoid, as the oocyte enlarges, may be relatively insensitive to a decline in long-range, anterior-directed forces because its requirements for such forces are less than those of the nucleus and gurken mRNA (Brendza, 2002).

Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes

Mass movements of cytoplasm, known as cytoplasmic streaming, occur in some large eukaryotic cells. In Drosophila oocytes there are two forms of microtubule-based streaming. Slow, poorly ordered streaming occurs during stages 8-10A, while pattern formation determinants such as oskar mRNA are being localized and anchored at specific sites on the cortex. Then fast well-ordered streaming begins during stage 10B, just before nurse cell cytoplasm is dumped into the oocyte. The plus-end-directed microtubule motor kinesin-1 is required for all streaming and is constitutively capable of driving fast streaming. Khc mutations reduce the velocity of kinesin-1 transport in vitro, block streaming, yet still support posterior localization of oskar mRNA -- this suggests that streaming is not essential for the oskar localization mechanism. Inhibitory antibodies indicated that the minus-end-directed motor dynein is required to prevent premature fast streaming, suggesting that slow streaming is the product of a novel dynein-kinesin competition. Since F-actin and some associated proteins are also required to prevent premature fast streaming, these observations support a model in which the actin cytoskeleton triggers the shift from slow to fast streaming by inhibiting dynein. This allows a cooperative self-amplifying loop of plus-end-directed organelle motion and parallel microtubule orientation that drives vigorous streaming currents and thorough mixing of oocyte and nurse-cell cytoplasm (Serbus, 2005).

To address questions about microtubule-based cytoplasmic streaming in Drosophila oocytes, functional disruption approaches were combined with fixed and time-lapse fluorescence microscopy. The results confirm that plus-end-directed kinesin-1 is the primary motor for both slow and fast streaming, and, furthermore, that it is constitutively capable of driving fast streaming. The minus-end-directed motor cytoplasmic dynein does not contribute force for fast streaming; rather, dynein and a normally regulated actin cytoskeleton impede the fast streaming activity of kinesin-1, allowing only slow streaming currents prior to stage 10B (Serbus, 2005).

It is reasonable to assume that the purpose of active but random transport processes like streaming is to facilitate the dispersal of cytoplasmic components that do not diffuse fast enough to support cellular and developmental demands. However, it could also be important for asymmetric localization processes by facilitating encounters of cytoplasmic components with localized anchors. More specific insights into how microtubule-based streaming contributes to particular processes have been elusive, in part because the only means to prevent streaming was to eliminate microtubules, which are needed for many fundamental cellular processes. Identification of kinesin-1 as the motor for streaming in Drosophila provides the opportunity for more focused studies, because kinesin-1 has a narrower range of functions and is not essential for early oocyte development (Serbus, 2005).

The Khc allelic series allowed investigation of the significance of nurse cell/ooplasm mixing. Khc-null oocytes, with no streaming, usually show yolk stratification as evidence of mixing failure. Embryos developing from those oocytes arrest in early stages, suggesting that mixing may be important for subsequent development. However, hypomorphic Khc17 oocytes, which support weak fast streaming in only one-third of oocytes, allow three-fourths of the derived embryos to develop to adulthood. Yolk stratification is not seen in Khc17 oocytes, suggesting that some mixing can occur without ordered streaming. Although these observations are consistent with the hypothesis that vigorous ooplasmic mixing helps optimize development, it is likely that fast streaming is not absolutely essential (Serbus, 2005).

The Khc allelic series also allowed exploration of a role for slow ooplasmic streaming in determinant mRNA localization. The null allele Khc27 prevents streaming: it blocks oskar mRNA accumulation at the posterior pole and it blocks gurken mRNA localization to the anterodorsal corner. However, the hypomorphic alleles Khc17 and Khc23, which prevented most slow streaming, support both oskar and gurken localization. Thus, although localization of both determinants requires Khc, it does not require slow streaming (Serbus, 2005).

It has been suggested that posterior oskar localization during stages 7-10a proceeds via two phases. (1) oskar RNPs are driven by kinesin-1 away from microtubule minus ends at the anterior and lateral cortex, which leads to a transient concentration of oskar in the central region of the oocyte. (2) Then diffusion or other random forces, coupled with a dearth of minus ends at the posterior cortex, facilitates encounters of oskar RNPs with posterior anchors. Tests of Khc17 and Khc23, which slow the ATPase activity and velocity of Khc in vitro, show a delay in the central accumulation of oskar, consistent with slowed kinesin-1-driven transport away from the anterolateral cortex. Strikingly, Khc17 and Khc23 allow that central accumulation to persist through later stages, as if the shift to posterior anchors is also slowed. This correlation between slowed motor mechanochemistry and slowed oskar localization supports the hypothesis that kinesin-1 links to and transports oskar RNPs in both phases of localization (Serbus, 2005).

If microtubules are poorly ordered during oskar localization, as suggested by GFP-tubulin imaging and by studies of fixed oocytes, how could kinesin-1 accomplish such directed posterior transport? There may be a special subset of microtubules, with plus-ends oriented directly toward the posterior pole, that are difficult to distinguish among a mass of randomly oriented microtubules. However, given that the period of oskar localization spans at least 10 hours, and that the distance from the oocyte center to the posterior pole is only 25-40 µm, such perfectly oriented transport tracks should not be necessary. With microtubule minus ends most abundant at the anterior cortex and least abundant at the posterior cortex, plus ends should be somewhat biased toward the posterior. If kinesin-1 binds an oskar RNP and transports it to a plus end, then binds a neighboring microtubule and runs to its plus end, and so forth, it would accomplish a biased random walk away from the anterolateral cortex that would concentrate oskar RNPs near posterior anchors. This highlights a central question about the mechanism of localization. What is the degree of directional bias for oskar RNP transport? Advances in osk RNP imaging that allow single particle tracking will be needed to obtain clear answers to that question (Serbus, 2005).

Regarding the mechanism of streaming, a model is suggested in which kinesin-1 drives plus-end-directed motion of cargoes that act as impellers, exerting force on ooplasm that surrounds them. Concerted movement of multiple impellers along neighboring microtubules that are oriented in the same general direction creates streams of ooplasm. Prior to stage 10B, small streams occur, but are slow and not well-ordered because dynein resists both plus-end-directed transport and parallel ordering of microtubules. This resistance may be accomplished via: (1) a tug-of-war between opposing motors co-attached to individual impellers; (2) by movement of different impellers in opposite directions, imparting conflicting forces on cytoplasm; or (3) competition by dynein and kinesin for the same binding site on microtubules. Regardless of how dynein interferes with kinesin-1, just before nurse cell cytoplasm is dumped into the oocyte, dynein is suppressed. This allows kinesin-1 to generate fast plus-end-directed impeller transport that sweeps microtubules into parallel arrays that then enhance more robust currents that enhance larger arrays, and so forth, in a self-amplifying loop (Serbus, 2005).

The finding that dynein inhibition enhances a kinesin-1-driven transport process provides the first direct indication of a competitive relationship between opposing microtubule motors. Other studies have produced convincing evidence of alternating coordination between dynein and plus-end-directed motors in a number of processes, including transport of Drosophila embryo lipid droplets, Drosophila cultured cell RNPs and peroxisomes, Drosophila axonal mitochondria (A. Pilling, PhD thesis, Indiana University, 2005, cited in Serbus, 2005), and Xenopus pigment granules. In those processes, inhibition of one motor does not enhance transport in the opposite direction. In fact kinesin-1 inhibition inhibits not only plus-end transport but also dynein-driven minus-end transport. Furthermore, dynein depletion can inhibit both directions of peroxisome transport, confirming that kinesin-1 and dynein each can have positive influences on the other. The observation of competition between dynein and kinesin-1 suggests that alternating coordination and positive interactions between microtubule motors are not a uniform rule, and that some processes have evolved to take advantage of motor competition (Serbus, 2005 and references therein).

If slow streaming is a product of kinesin-dynein competition, why does Khc inhibition arrest all streaming, rather than freeing dynein to drive reverse streaming? One possibility is that although forces from impeller-bound dynein can resist kinesin-1 and confound parallel microtubule ordering, it is not sufficiently processive to generate minus-end-directed streaming currents. A second possibility is that Khc inhibition blocks minus-end as well as plus-end-directed streaming forces, similar to the processes noted above in which dynein transport activity is dependent on Khc (Serbus, 2005).

The observation that actin cytoskeleton depolymerization or mutation of certain actin-interacting proteins can induce premature kinesin-1-driven fast streaming is particularly interesting. Actin filaments are most abundant in the cortex and ring canals of the oocyte and nurse cells, but filaments probably also traverse the internal cytoplasm. An intact actin cytoskeleton could physically assist dynein in resisting kinesin-based plus-end-directed transport during slow streaming, either passively by increasing viscosity or actively by generating antagonistic forces. The active force idea is supported by reports that myosin V can alter the balance between alternating dynein and kinesin-2-driven runs of melanosomes in Xenopus. Drosophila myosin V inhibition tests have not yet been reported, but a disordered cortical actin cytoskeleton in Moesin mutant oocytes does not trigger premature fast streaming, suggesting that well-ordered actin-based forces may not be important for the streaming control mechanism. An alternative to such physical resistance is that dynein inhibitory factors are sequestered by F-actin prior to stage 10B. Then, just before dumping, those factors are released, dynein is inhibited, and kinesin-1 is freed to drive fast streaming (Serbus, 2005).

Recently, several other factors have been identified that are required for prevention of premature fast streaming. Mutations in Maelstrom (Mael), Orb and Spindle-E (Spn-E) allow premature fast streaming and parallel microtubule arrays during stages 8-10A. Orb, a CPEB homolog, is required for osk translation, spn-E is an RNA helicase, and Mael is a modifier of Vasa, which is another RNA helicase. Perhaps these proteins control expression of actin regulators or other factors needed to prevent premature activation of a dynein inhibitory signal. Future work aimed at identifying the regulatory mechanisms that control kinesin in oocytes should be an important focus in understanding the slow-fast streaming transition and also for the broader issue of how the functions of the actin and microtubule cytoskeletons are integrated (Serbus, 2005 and references therein).

Milton, which has been shown to associate with Kinesin and to mediate axonal transport of mitochondria, controls the early acquisition of mitochondria by Drosophila oocytes

Mitochondria in many species enter the young oocyte en mass from interconnected germ cells to generate the large aggregate known as the Balbiani body. Organelles and germ plasm components frequently associate with this structure. Balbiani body mitochondria are thought to populate the germ line, ensuring that their genomes will be inherited preferentially. Milton, a gene whose product has been shown to associate with Kinesin and to mediate axonal transport of mitochondria, is needed to form a normal Balbiani body. In addition, germ cells mutant for some milton or Kinesin heavy chain (Khc) alleles transport mitochondria to the oocyte prematurely and excessively, without disturbing Balbiani body-associated components. These observations show that the oocyte acquires the majority of its mitochondria by competitive bidirectional transport along microtubules mediated by the Milton adaptor. These experiments provide a molecular explanation for Balbiani body formation and, surprisingly, show that viable fertile offspring can be obtained from eggs in which the normal program of mitochondrial acquisition has been severely perturbed (Cox, 2006).

The Balbiani body, a large aggregate of mitochondria frequently associated with other membranous organelles and germ plasm components, is found in the newly formed oocytes of diverse species. Although it has been postulated to play a role in germ cell development and mitochondrial inheritance, no function for the Balbiani body has been demonstrated. Previously, the Drosophila Balbiani body arises when a large number of mitochondria from sister germ cells associate with the fusome, move towards its center and enter the oocyte en masse where they supplement the pre-existing mitochondria of the oocyte (Cox, 2003). Like ooctye development itself, Balbiani body formation requires the genes hts and egl, suggesting that mitochondrial movement depends on Dynein/dynactin-mediated minus-end directed transport along polarized microtubules (Cox, 2003). Studies of the mitochondrial adaptor protein Milton and its partner Kinesin heavy chain now show that plus-end directed mitochondrial transport determines when and how large a Balbiani body will form (Cox, 2006).

Mitochondrial position within cells of diverse types is frequently regulated by motor-dependent transport along microtubules. Often such positioning optimizes the ability of mitochondria to generate energy or metabolic products in appropriate subcellular locations. In Drosophila photoreceptors, neurons and in cultured cells, Milton plays a key role in positioning mitochondria by acting as a adaptor molecule between mitochondria and the Khc plus-end-directed microtubule motor. Null Khc mutations and type II milt alleles cause premature entry of an excess number of mitochondria into the oocyte. This suggests that the orchestrated movement of mitochondria within germline cysts and its sudden entry into the oocyte during follicle formation is controlled by plus-end directed transport machinery that opposes Dynein-mediated minus-end directed movement towards the oocyte. Plus-end directed activity is not needed for mitochondria to associate with the fusome; normal fusome interactions are still observed in the absence of Khc or milt function. However, the opposing action of Milt and Khc appears to be particularly effective near ring canals, especially the four oocyte ring canals, just outside of which mitochondria accumulate for a period of 1-2 days prior to follicle formation. As a new follicle prepares to bud off, an unknown modulation relieves the standoff and leads to the rapid influx of mitochondria into the oocyte where they coalesce with endogenous mitochondria to form the Balbiani body. In the absence of any movement, as in milt92 cysts, or in cysts with compromised Dhc function, a much smaller cluster of mitochondria forms in the oocyte, made up only of organelles inherited during germ cell divisions (Cox, 2006).

Complete loss of Milton did not enhance mitochondrial movement into the oocyte, as expected if its sole function was linkage to Khc. Instead, milt is needed for both minus-end directed and plus-end directed movement. Upregulation of Milt-PB relative to Milt-PA favors Dynein-based movement, but the basis for this effect remains unclear. Both Milt isoforms contain a common Kinesin binding domain, and associate with Kinesin in vivo. Evidence that Milt proteins bind Dynein directly is lacking, and the related GRIF1 protein does not bind Dynein. Thus, Milt-PB probably promotes linkage of mitochondria to Dynein indirectly, perhaps by binding and modulating Dynactin. Consistent with this view, the HAP-1 domain that differs between the two isoforms has been predicted to mediate interaction with Dynactin. Thus, changes in the relative amounts of Milt isoforms, and in their interactions with mitochondria appear to regulate the location of these organelles (Cox, 2006).

Related mechanisms may control the movement along the fusome and entry into the oocyte of other cargos besides mitochondria. Organelles such as Golgi elements, and specific mRNAs such as Bic-D, oskar and cup localize towards the center of developing 16-cell cysts, and enter the oocyte (Cox, 2003). oskar and cup RNA transiently associate with the Balbiani body in forming follicles (Cox, 2003). However, all these RNAs localize to the initial cyst cell earlier than mitochondria (Cox, 2003), and it is found that Cup continues to accumulate preferentially in the oocyte even in Dhc64C6-6/6-12 mutants that block mitochondrial transport. Consequently, even if all these components are localized based an interplay of plus-end- and minus-end-directed micotubule transport, their movement towards the oocyte is regulated differently, possibly because each is linked by cargo-specific adaptors (Cox, 2006).

Finally, these experiments provide the first test of Balbiani body function. The initial wave of mitochondria that enter the oocyte of new follicles in the Balbiani body have been proposed to have high fitness, and to represent the inheritance bottleneck of mitochondrial genomes (Cox, 2003). Oocytes from milt alleles, where this process has been strongly disrupted, still give rise to viable and fertile offspring. In part, this may be due to the observation that an independent system of mitochondrial copy number control acts to correct initial increases or deficits in oocyte mitochondrial number. Future studies will be required to determine if mitochondrial inheritance patterns are altered in milt class II mutants, and if the offspring of these alleles suffer an increased incidence of mitochondrial dysfunction over their lifespan (Cox, 2006).

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. This study report a careful analysis of the oocyte cytoskeleton. Thick actin bundles are identified at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, the data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. Actin bundle formation requires Profilin but not Capu and Spire. Thus, the results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, the data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. The release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent (Wang, 2008).

This study reports the existence of actin bundles at the cortex of the oocyte that are involved in the cortical localisation of γTubulin. γTubulin is part of the γTubulin ring complex that is stabilising the minus ends of MTs. The presence of γTubulin alone does not allow to distinguish whether the protein is part of a microtubule organising centre (MTOC) that nucleates MTs or whether it only protects existing MTs from depolymerisation. This study used γTubulin solely as a marker for the MT minus ends; these are embedded within the cortical actin bundles before stage 10b (Wang, 2008).

The cytoskeletal rearrangements at stage 10b include the disassembly of the cortical actin bundles, the redistribution of the MT minus ends from the cortex to subcortical regions and the formation of MT arrays parallel to the oocyte cortex. Concomitantly with these cytoskeletal changes, the transition from slow to fast cytoplasmic streaming is triggered. What is the causal relationship between these events? The finding that interfering with actin bundle formation by drug treatment and GFPactin5c overexpression results in MT minus ends redistribution, MT array formation and premature fast streaming indicates that actin bundling acts upstream of MT reorganisation and streaming. The analysis of Khc mutants allows to further dissect the subsequent steps reorganising the MT cytoskeleton. In the absence of streaming, caused by the loss of Khc function, the redistribution of MT minus ends occurs normally, while the formation of MT arrays is abolished. Thus, minus end redistribution is upstream of streaming, and array formation is downstream. It is therefore proposed that streaming is initiated by the disassembly of the cortical actin bundles resulting in loss of cortical MT minus end anchoring. It is further proposed that the redistribution of the minus ends to subcortical regions is important for the reorganisation of the MT cytoskeleton into arrays that run parallel to the oocyte cortex. At this step a previously suggested self amplifying loop could be initiated, in which MT array formation and Kinesin movement enhance each other. In this loop the Kinesin driven streaming helps to sweep MTs into parallel arrays, which in turn allow more robust currents in the cytoplasm (Wang, 2008).

How do the actin binding proteins Capu, Spire and Profilin act on the oocyte cytoskeleton to prevent premature cytoplasmic streaming? chic/Profilin mutants and latrunculin A treatment both interfere with bundle formation. Latrunculin A treatment inhibits actin polymerisation by binding to and sequestering actin monomers. Profilin is involved in actin polymerisation by delivering actin monomers to the growing ends of actin filaments. Thus, latrunculin A and Profilin mutants appear to interfere with bundling by limiting the pool of monomers that can be added to growing actin filaments. In contrast, capu and spire mutants are not required for the formation of actin bundles. It is proposed that Capu and Spire anchor the MT minus ends in a scaffold provided by the cortical actin bundles. The lack of Capu and Spire activity in the mutants prevents cortical MT anchoring and allows streaming in the presence of actin bundles. This model is supported by the work that has shown that Capu and Spire proteins are able to crosslink F-actin and MTs, and that both proteins localise to the oocyte cortex (Wang, 2008).

The regulation of fast ooplasmic streaming could be controlled at the level of the cortical localisation of Capu and Spire. The displacement of the two proteins from the cortex at stage 10b might result in loss of MT minus end anchoring, and thereby induce fast streaming. To test this the localisation of GFP-Capu and GFP-Spire was analysed in cross sections of oocytes. However, no difference was detected in the localisation of the two proteins before and after onset of fast streaming. In addition, no displacement of GFP-Capu and GFP-Spire was detected after induction of premature streaming by latrunculin A treatment. Thus, Capu and Spire activities are not regulated at the level of their localisation (Wang, 2008).

A different mode of Capu and Spire regulation is suggested by their genetic and biochemical interaction with Rho1. This interaction has led to a model in which Rho1 initiates fast streaming by regulating the crosslinking activities of Capu and Spire. This study shows that the prevention of streaming requires not only capu and spire but also the presence of actin bundles. The formation of these bundles occurs, however, independently of capu and spire. This suggests that the onset of fast streaming is not only controlled by regulating Capu and Spire activities, but also by disassembly of the actin bundles (Wang, 2008).

Genes that are involved in actin regulation in the oocyte were also tested but these do not induce premature streaming. For capulet, swallow and moesin mutants the formation of ectopic actin clumps has been reported reflecting defects in the organisation of the oocyte actin cytoskeleton. The presence of ectopic F-actin in the oocyte cytoplasm was confirmed in these mutants, but nevertheless the formation cortical actin bundles was detected. Thus, actin defects in the oocyte do not necessarily affect cortical actin bundling (Wang, 2008).

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought (Kucherenko, 2008).

The screens described in this paper revealed some expected interactors, Dys, Cam and Khc. Calmodulin, a calcium binding protein required for muscle and neuronal functions has previously been shown to interact with mammalian the Dg-Dys complex. However, whether reduction of Cam activities suppresses or enhances the muscular dystrophy phenotype is not totally clear. Targeted inhibition of Cam signaling exacerbates the dystrophic phenotype in mdx mouse muscle while genetic disruption of Calcineurin improves skeletal muscle pathology and cardiac disease in ä-sarcoglycan null mice. Since reduction of Cam showed suppression of the phenotypes caused by reduction of the long forms of dystrophin in the Drosophila wing, it will be interesting to analyze whether reduction of Cam will suppress the Drosophila Dys muscle phenotype as well. Khc involvement in Dg-Dys complex is also expected since work in mammalian system has shown that Khc can bind Dystrobrevin, a component of Dg-Dys complex. It will be interesting to test in the future whether Drosophila Dystrobrevin can similarly bind Khc and what the functional significance of this interaction is in muscles and neurons. In oocyte development Khc is required as early as is Dys and Dg. It is, therefore, interesting to test the potential requirement of dystrobrevin in this process and to further dissect the Khc function in this complex during early polarity formation (Kucherenko, 2008).

Therefore, interactions were found with Khc, Lis-1 and Dmn, three genes known to be part of the Dynein-Dynactin complex which in addition to Kinesin microtubule motor activity have been shown to be necessary for establishment of intracellular polarity within the Drosophila oocyte. In mid-oogenesis dynein, dynactin and kinesin are thought to act cooperatively in cargo transport. Since these genes interact with Dys and show similar phenotypes in Orb localization, it will be interesting to dissect their potential functional interactions with Dys in early oocyte development. Furthermore, since mammalian Dystrobrevin physically interacts with Khc, it is plausible, that the Dynein-, Dynactin-, Kinesin-complex will utilize localization cues set-up by Dg-Dys Complex (Kucherenko, 2008).

By screening for alterations of a dominant wing vein phenotype modifiers of the DGC were found that are involved in cytoskeletal organization. Initial characterization of some of these genes revealed that they have phenotypes also in other tissues, in which the DGC is known to function. These tissue/cell types include the oocyte, the brain and the indirect flight muscles. This argues strongly that the identified interactors may be involved globally in DGC function. Further study is required to determine mechanistically how these modifiers work in the context of the Dg-Dys complex. However a common theme, already arising is that the identified interactors appear to regulate cytoskeletal rearrangement. Mechanistic understanding of how the new interactors might regulate Dg-Dys communication with cytoskeleton of muscle cells may serve as a basis for the development of novel therapeutic approaches that might improve the quality of life of individuals afflicted with muscular dystrophy (Kucherenko, 2008).

Spatial control of branching within dendritic arbors by dynein-dependent transport of Rab5-endosomes

Dendrites allow neurons to integrate sensory or snaptic inputs, and the spatial disposition and local density of branches within the dendritic arbor limit the number and type of inputs. Drosophila dendritic arborization (da) neurons provide a model system to study the genetic programs underlying such geometry in vivo. This study reports that mutations of motor-protein genes, including a dynein subunit gene (dlic) and kinesin heavy chain (khc), caused not only downsizing of the overall arbor, but also a marked shift of branching activity to the proximal area within the arbor. This phenotype is suppressed when dominant-negative Rab5 is expressed in the mutant neurons, which deposit early endosomes in the cell body. It was also shown that in dendritic branches of the wild-type neurons, Rab5-containing early endosomes are dynamically transported. When Rab5 function alone is abrogated, terminal branches were almost totally deleted. These results reveal an important link between microtubule motors and endosomes in dendrite morphogenesis (Satoh, 2008).


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Kinesin heavy chain: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 April 2015

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