Kinesin heavy chain

DEVELOPMENTAL BIOLOGY

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 (Dhc64C^6-6/Dhc64C^6-12) 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).

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 cpb^M143. In these cpb^M143 mosaic animals, the nuclear regions of many photoreceptors were observed in the optic stalk and brain (Whited, 2004).

To confirm that the cpb^M143 mutant photoreceptor defect was due to a loss of cpb function, an additional strong loss-of-function cpb allele, cpb^F44, 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 cpb^F44 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];cpb^M143/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 cpb^M143/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 (Glued^DN) that resembles the protein product of Glued¹. Glued^DN 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 Glued^DN 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 Glued^DN 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, khc^k13219 and khc^k13314, 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 Glass_38-1 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. Glass_38-1:Glued^DN animals contained an average of 11±1 photoreceptor nuclei within the optic stalk. However, Glass_38-1:Glued^DN animals heterozygous for either khc^k13314 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 Glued^DN 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 Glued^DN 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:Glued^DN animals had no neuronal nuclei in this region, Glass_38-1:Glued^DN animals contained 7±1. A reduction of khc gene dosage in Glass_38-1:Glued^DN; khc^k13314/+ and Glass_38-1:Glued^DN; 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).

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 K_{0.5, 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 k_cat (5.9 versus 20 s^-1 for wild-type), k_cat/K_m,ATP (0.065 versus 0.2 µM^-1 s^-1), and k_cat/K_0.5,MT (3.6 versus 25 µM^-1 s^-1), yet both the K_m,ATP and K_0.5,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 Khc^null) was used to generate single Khc^null/Khc^null cells in Khc^null/Khc⁺ larvae. The proliferative capacities of single Khc^null/Khc^null 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 Khc^null 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 Khc^null 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. Khc^null 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, Khc^null 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 Khc^null 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 Dhc64C^6-6/6-12 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).

REFERENCES

Batut, J., Howell, M. and Hill, C. S. (2007). Kinesin-mediated transport of Smad2 is required for signaling in response to TGF-beta ligands. Dev. Cell 12(2): 261-74. Medline abstract: 17276343

Bi, G. Q., et al. (1997). Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis. J. Cell Biol. 138: 999-1008. 9281579

Bonini, N.M. (2002). Chaperoning brain degeneration. Proc. Natl. Acad. Sci. 99: (Suppl 4) 16407-16411. 12149445

Bowman, A. B., et al. (2000). Kinesin-dependent axonal transport is mediated by the sunday driver (SYD) protein. Cell 103: 583-94. 11106729

Brendza, K. M., et al. (1999). Lethal kinesin mutations reveal amino acids important for atpase activation and structural coupling. J Biol Chem, Vol. 274: 31506-31514. 10531353

Brendza, K. M., et al. (2000). A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor. J. Biol. Chem. 275: 22187-22195. 10767290

Brendza, R. P., et al. (2000a). Clonal tests of conventional kinesin function during cell proliferation and differentiation. Mol. Biol. Cell 11(4): 1329-43. 10749933

Brendza, R. P., et al. (2000b). A function for kinesin I in the posterior transport of Oskar mRNA and Staufen protein. Science 289(5487): 2120-2. 11000113

Brendza, R. P., et al. (2002). Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes. Current Biology 12: 1541-1545. 12225672

Cai, Q., Gerwin, C. and Sheng. Z. H. (2005). Syntabulin-mediated anterograde transport of mitochondria along neuronal processes. J. Cell Biol. 170: 959-969. 16157705

Clark, I., et al. (1994). Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr Biol 4: 289-300.

Cox, R. T. and Spradling, A. C. (2003). A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130: 1579-1590. Medline abstract: 12620983

Cox, R. T. and Spradling, A. C. (2006). Milton controls the early acquisition of mitochondria by Drosophila oocytes. Development 133(17): 3371-7. Medline abstract: 16887820

Dahlgaard, K., Raposo, A. A., Niccoli, T. and St Johnston, D. (2007). Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte. Dev. Cell 13(4): 539-53. PubMed citation: 17925229

Duncan, J. E. and Warrior, R. (2002). The cytoplasmic Dynein and Kinesin motors have interdependent roles in patterning the Drosophila oocyte. Curr. Biol. 12: 1982-1991. 12477386

Gho, M., McDonald, K., Ganetzky, B. and Saxton, W. M. (1992). Effects of kinesin mutations on neuronal functions. Science 258: 313-316. 1384131

Gindhart, J. G., Desai, C. J., Beushausen, S., Zinn, K., and Goldstein, L. S. B. (1998). Kinesin light chains are essential for axonal transport in Drosophila. J. Cell Biol. 141: 443-454. 9548722

Glater, E. E., Megeath, L. J., Stowers, R. S. and Schwarz, T. L. (2006). Axonal transport of mitochondria requires Milton to recruit kinesin heavy chain and is light chain independent. J. Cell Biol. 173(4): 545-57. 16717129

Goldstein, L .S. B. and Philp, A. V. (1999). The road less traveled: emerging principles of kinesin motor utilization. Annu. Rev. Cell Dev. Biol. 15: 141-183. 10611960

Goldstein, L. S. B. and Yang, Z. (2000). Microtubule-based transport systems in neurons: the roles of kinesins and dyneins. Annu. Rev. Neurosci. 23: 39-72

Goodson, H.V., Valetti, C. and Kreis, T.E. (1997). Motors and membrane traffic. Curr. Opin. Cell Biol. 9: 18-28. 9013678

Gorska-Andrzejak, J., Stowers, R. S., Borycz, J., Kostyleva, R., Schwarz, T. L. and Meinertzhagen, I. A. (2003). Mitochondria are redistributed in Drosophila photoreceptors lacking milton, a kinesin-associated protein. J. Comp. Neurol. 463(4): 372-88. 12836173

Gunawardena, S. and Goldstein, L. S. B. (2001). Disruption of axonal transport and neuronal viability by Amyloid precursor protein mutations in Drosophila. Neuron 32: 389-401. 11709151

Gunawardena, S., et al. (2003). Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron 40: 25-40. 14527431

Guo, X., et al. (2005). The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses. Neuron. 47: 379-393. 16055062

Hopmann, R. and Miller, K. G. (2003). A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle. Mol. Biol. Cell 14: 118-128. 12529431

Horiuchi, D., Barkus, R. V., Pilling, A. D., Gassman, A. and Saxton, W. M. (2005). APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila. Curr. Biol. 15(23): 2137-41. 16332540

Horiuchi, D., Collins, C. A., Bhat, P., Barkus, R. V., Diantonio, A. and Saxton, W. M. (2007). Control of a kinesin-cargo linkage mechanism by JNK pathway kinases. Curr. Biol. 17(15): 1313-7. Medline abstract: 17658258

Huang, T. G. and Hackney, D. D. (1994a). Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization. J Biol Chem 269: 16493-16501. 8206959

Huang, T. G., Suhan, J. and Hackney, D. D. (1994b). Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli. J Biol Chem 269: 16502-7. 8206960

Hurd, D. D., Stern, M. and Saxton, W. M. (1996a). Mutation of the axonal transport motor kinesin enhances paralytic and suppresses Shaker in Drosophila. Genetics 142: 195-204. 8770597

Hurd, D. D. and Saxton, W. S. (1996b). Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport