Gene name - Bicaudal D
Cytological map position - 36C2-36C11
Function - presumed cytoskeletal element
Symbol - BicD
FlyBase ID: FBgn0000183
Genetic map position - 2-52.9
Classification - alpha helical coiled coil protein
Cellular location - cytoplasmic
In normal flies each of the three thoracic and eight abdominal segments has a characteristic belt of hairs (denticles); the hairs in the abdominal segments generally are thicker, and hence their denticle belts are more readily apparent. Mutation of BicD results in a bicaudal phenotype: in the bicaudal embryo, the head, thorax, and anterior-most three to five abdominal segments are replaced by a mirror image of the posterior abdominal segments and terminalia, including the posterior spiracles (Suter, 1989, Wharton, 1989 and Mohler, 1986).
Earlier literature on Bicaudal-D provides an impressive number of explanations for the origin of bicaudality. One explanation suggests that Bic-D has a direct role in the localization of the posterior determinant nanos (Wharton, 1989). Equally likely was the belief that Bic-D regulates the anterior localization of Bicoid mRNA (Suter, 1989). A third idea was that Bic-D is required for the determination of the one cystocyte to become the oocyte (the egg has a total of 16, 15 of which become nurse cells). This determination is not made in BicD mutants, thereby preventing the posterior migration of the oocyte (Ran, 1994). However, over time it has become clear that establishment of oocyte identity is a multistage process involving multiple factors and events and that BicD regulates multiple aspects of this process. BicD is indeed involved in establishing the developmental fate of the the oocyte as distinct from that of nurse cells, and a failure of this process interfers with the localization of anterior and posterior determinants of oocyte polarity. A major aspect of the determination of developmental fate of the oocyte is, in fact, localization of various determinants in the presumptive oocyte, and in this process, Bic-D seems to play an important role. These localization events take place during mid-oogenesis, after the establishment of oocyte fate. BicD plays a role in early fate determination of the oocyte and in subsequent localization of the mRNA determinants of oocyte polarity (Mach, 1997 and Swan, 1996).
Three proteins have been implicated in oocyte determination: Stonewall, a presumptive transcription factor, and BicD and Egalitarian, both with novel sequences. The microtubule cytoskeleton is an additional element in oocyte determination. Treating wild-type flies with microtubule-depolymerizing drugs such as colchicine also causes a 16-nurse-cell phenotype, implicating microtubule structure as an important component of the oocyte determination mechanism. A microtubule organizing center (MTOC) forms in the presumptive oocyte just after the formation of the 16-cell cluster; in Bic-D mutants this MOTC does not form (Theurkauf, 1993). Because BicD protein localizes to a single cell in Bic-D mutants, BicD can localize without the formation of the MOTC (Mach, 1997).
The beginnings of determination of oocyte fate precede the function of Bic-D. In fact, the oocyte fate is determined earlier than the 16 cell stage. A bias toward the oocyte fate already may be set during the first division of the oocyte precursor, or cystoblast, when one daughter inherits the spectrosome, a spectrin-rich organelle that seems to play a role in orienting the plane of division of the cystoblast progeny. Of particular interest is the association of spectrosomes with the pole of the mitotic spindle. During the first cystoblast division, spectrosome material is associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing spectrosome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed (Lin, 1995).
What then is the role of Bic-D in establishing oocyte fate? The recent characterization of the egalitarian gene has provided new insight into this question. Like BicD, Egalitarian is a novel protein that colocalizes with BicD protein at all stages of oogenesis. Egl and BicD proteins localize to the oocyte in three stages that correlate with the stepwise polarization of the oocyte. From stage 2 until stage 7 of oogenesis, EGL mRNA is concentrated at the posterior cortex of the oocyte. The distribution of Egl protein strikingly resembles the localization of the minus end of microtubules (see ß1 tubulin for more information). During stages 1-6 of oogenesis, a microtubule network extends from an MTOC located at the posterior cortex of the early oocyte into the nurse cells. During stage 8 of oogenesis the microtubule network repolarizes and the microtubules orient from the anterior cortex (Theurkauf, 1992). At this time EGL transcript localizes in an anterior ring at the nurse cell-oocyte boundary. This distribution is similar to that of other RNAs, such as K10, oo18 RNA-binding protein (orb), BicD, and oskar, all of which accumulate early in the oocyte and sometime later form a transient anterior ring. By stage 10 of oogenesis, EGL mRNA is distributed evenly throughout the oocyte but persists in the oocyte into early embryogenesis (Mach, 1997).
EGL protein is detectable in the germarium and initially is distributed evenly within the newly formed 16-cell cyst. Once the cyst flattens in germarial region 2B, the protein is often concentrated in the two cells that have four canals. By stage 1 of oogenesis, Egl protein localizes to a single cell, the future oocyte. From stage 2 to stage 7 of oogenesis, Egl protein is enriched at the posterior cortex of the oocyte. At stage 8 of oogenesis, Egl protein shifts to the anterior cortex in a ring around the margin of the oocyte, where the oocyte, nurse cells, and follicle cells meet. Disrupting the microtubule network with colchicine abolishes EGL localization in the oocyte. Thus, EGL localization requires microtubule organization. Morover, it has been shown (Theurkauf, 1993) that in egl and BicD mutants the microtubule network, although established initially, is not maintained. It is therefore likely that the effect of microtubules on oocyte determination is mediated by EGL and BicD, and that these two proteins in turn reinforce or maintain the microtubule network after an initial polarity has been established. There is no hierarchical relationship between EGL and BicD localization; instead, localization of each requires the other (Mach, 1997).
Experiments with antibodies to EGL and BicD make it clear that there is a physical interaction between the two proteins. Since mutation of either protein results in the loss of oocyte identity with the consequent adoption of nurse cell fate by all 16 cystocytes, both proteins play a role in oocyte determination. They are also required to establish oocyte polarity. Females carrying a dominant BicD mutation produce embryos that develop with reduced head structures attributable to the partial mislocalization of OSK mRNA to the anterior of the oocyte (Ephrussi, 1991). Antibody staining reveals that compared with wild type, BicD protein accumulation in the early oocyte is more pronounced in BicD-Dominant mutants and that the protein remains at the anterior pole of mutant oocytes during later stages of oogenesis and early embryogenesis (Wharton, 1989).
To determine whether egl and Bic-D directly affects the extent to which OSK mRNA mislocalizes in embryos, the distribution of OSK mRNA was examined in BicD-Dominant mutants. Reducing the amount of egl wild-type product decreases ectopic localization of osk to the anterior and increasing the amount of egl wild-type product enhances the mislocalization of OSK to the anterior. Because the effect of BicD-Dominant mutants depends on egl wild type function, it is concluded that egl and BicD act in the same pathway and that the two function in concert to control OSK mRNA localization. It is also thought that Egl and BicD have a role in dorsoventral polarity, as mutation of the two genes reduce the level of GURKEN mRNA. Localization of GUR is also known to require an intact microtubule cytoskeleton (Mach, 1997).
Each step - first determination of oocyte fate, then specification of the anterior-posterior axis, and finally specification of the dorsoventral axis - requires both RNA transport along a polarized microtubule network and the function of the EGL-BicD complex. The distribution of the two proteins resembles that of the minus ends of microtubules, and mutations in either disrupt microtubule stability. Although it is still possible that the EGL-BicD complex affects RNA localization solely by stabilizing microtubule structure, it is likely that the complex also acts as a link between microtubules and the RNA localization machinery. If the two proteins act directly to localize RNAs, these proteins may either bind RNA or associate with an RNA-binding protein such as Orb, whose distribution is strikingly similarly to that of Egl and BicD, forming a pattern dependent on Egl and BicD function (Mach, 1997, Lantz, 1994 and Christerson, 1994)
Meiosis is a specialized cell cycle limited to the gametes in Metazoa. In Drosophila, oocyte determination and meiosis control are interdependent processes, and BicD appears to play a key role in both. However, the exact mechanism of how BicD-dependent polarized transport could influence meiosis and vice versa remains an open question. This article reports that the cell cycle regulatory kinase Polo binds to BicD protein during oogenesis. Polo is expressed in all cells during cyst formation before specifically localizing to the oocyte. This is the earliest known example of asymmetric localization of a cell-cycle regulator in this process. This localization is dependent on BicD and the Dynein complex. Loss- and gain-of-function experiments showed that Polo has two independent functions. On the one hand, it acts as a trigger for meiosis. On the other, it is independently required, in a cell-autonomous manner, for the activation of BicD-dependent transport. Moreover, Polo overexpression can rescue a hypomorphic mutation of BicD by restoring its localization and its function, suggesting that the requirement for Polo in polarized transport acts through regulation of BicD. Taken together, these data indicate the existence of a positive feedback loop between BicD and Polo, and it is proposed that this loop represents a functional link between oocyte specification and the control of meiosis (Mirouse, 2006).
This paper describes the localization of the Polo protein and its genetic control in the Drosophila germline during early oogenesis. Polo has a peculiar subcellular localization in cytoplasmic dots that do not correspond to any well-known structures of germline cysts or to microtubule minus-ends where BicD accumulates. Polo has previously been described as colocalizing with several subcellular structures depending on cell cycle phase, but none of these corresponds to the localization observed in this study. Similar cytoplasmic dots were observed in the primordial germline cells of the Drosophila embryo as soon as they were formed, suggesting that this unusual localization could be a specific feature of the germline (Mirouse, 2006).
From region 2a onward, Polo dots are present mostly in the cells containing SCs. This is the first report of a cell-cycle regulator whose localization is spatially and temporally correlated with meiotic progression during early oogenesis. Moreover this correlation is still conserved in mutants that affect polarized transport and the restriction and maintenance of meiosis. This indicates that Polo localization is dependent on polarized transport. One possibility is that Polo itself is directly transported to the oocyte. This hypothesis is reinforced by the physical interaction between BicD and Polo proteins, according to the proposed function of BicD as adapter for Dynein cargos. However, the BicD-dependent localization of Polo is not sufficient to explain its expression profile. Polo is strongly expressed in region 1 of the germarium, and the overall amount of the protein in the cyst progressively decreases, becoming undetectable after stage 2. This degradation seems to be compensated in meiotic cells and then in the oocyte by the polarized transport. The progressive degradation of Polo is also observed in egl and BicD null mutants. Degradation in association with a complete absence of Polo transport may explain why all the cells of a cyst enter into meiosis in these mutants (all the cells contain the same amount of Polo), and then exit meiosis simultaneously (none of the cells preferentially accumulates enough Polo). Alternatively, rather than by direct transport of Polo to the oocyte, its asymmetric distribution in the cyst could be due to a differential control of its stability between nurse cells and oocyte under the control of the BicD-dependent polarized transport (Mirouse, 2006).
BicD and egl null mutants showed a very similar phenotype, in which all 16 cells of a cyst first enter into meiosis but subsequently lose the synaptonemal complexes (SCs). This phenotype cannot be compared with null mutants of the dhc, since Dynein is required at earlier steps of cyst formation. The human homolog of BicD interacts directly with Dynamitin, and this interaction is thought to mediate the interaction of BicD with the Dynein complex. In contrast to BicD, Dynamitin is not involved in the initial restriction of meiosis, showing that the interaction of BicD with Dynamitin, and thus probably Dynein, is not required for the initial restriction of meiosis. In a similar way, LC8 (cut up) null mutants or egl mutants that specifically block the interaction between Egl and LC8 do not interfere with the initiation of meiosis in only four cells. Transport of the BicD protein between the cyst cells is apparently not required for this first step, sinc the BicDPA66 allele or drug-induced microtubule depolymerization does not affect this initial restriction, although BicD is diffuse throughout the entire cyst. Finally, a null mutant for the plakin shot, which has been proposed to be an essential upstream component of the Dynein function in centrosome migration, exhibits variable meiotic phenotypes but allows a normal initial restriction of meiosis to four cells. These data are consistent with a function of BicD and Egl independent of Dynein in the initial restriction of meiosis (Mirouse, 2006).
Polo is involved in many crucial steps of the cell cycle, including the G2/M transition of mitosis and meiosis processes. This study shows that hypomorphic polo alleles lead to a delay in meiotic entry and that Polo overexpression can trigger meiosis in more than four cells per cyst in region 2a. These phenotypes could be related to the function of Polo in the G2/M transition. In vertebrates, Polo is an activator of the String/CDC25 phosphatase, and it has also been proposed that Polo can repress the kinases Myt1 and Wee1. String is the main activator of the cyclinB/CDC2 complex, the activity of which triggers the G2/M transition, whereas Myt1 and Wee are repressors of this complex. However, the role of the cyclin B and CDC25 in meiosis in Drosophila oogenesis is not yet well understood because, for example, CDC25 seems to act as a negative regulator of meiotic oocyte cell fate. Further investigations will be needed to determine how Polo triggers meiotic entry during early oogenesis (Mirouse, 2006).
This study has shown that in mutants with partial loss of polo function, SCs start to disassemble in region 3 but are well formed again in stage 2/3 before disappearing in the following stages. One possible hypothesis to explain how meiosis is finally properly maintained in polo hypomorphic mutants is that the repression of cyclin E by Dacapo during stage 2/3 represses endoreplication, and thus allows meiotic progression. This is consistent with the finding that the specific localization of Dacapo to the oocyte and its requirement for meiosis maintenance begins only in region 3. Moreover, null mutations of dacapo do not lead to a fully penetrant 16-nurse-cell phenotype, confirming the existence of a partially redundant control. Therefore, it is proposed that the balance in favor of meiosis is initially due to the localized activation of meiosis by Polo, and later to the localized inhibition of endoreplication by Dacapo, and that both mechanisms partially overlap (Mirouse, 2006).
It was also observed that Polo is required for the normal restriction of meiosis. Moreover, the defects in the restriction of meiosis caused by both loss and gain of polo function are correlated with defects in oocyte determination. Meiosis restriction and oocyte specification both depend on the Dynein complex and the BicD polarized transport system. Thus, it is assumed that these Polo phenotypes indicate that Polo is involved in polarized transport. This role may be indirect and thus reveals the influence of meiosis and cell-cycle control on oocyte differentiation. Such influence has been observed in situations where there is activation of the meiotic checkpoint due to a failure in DNA double-stand break repair. However, at least two results argue for a direct role of Polo in polarized transport, independently of its meiotic function. First, in mosaic germline cysts, nonmeiotic cells mutant for polo retain BicD protein. Thus, this phenotype cannot be due to the activation of the meiotic checkpoint. This strongly suggests that Polo is required in each cell of the cyst to initiate BicD-dependent transport to the presumptive oocyte. Second, the overexpression of Polo is able to restore the localization and therefore the function of BicDPA66 protein. Interestingly, this mutant allele is due to a single amino acid substitution (A40V) that leads to a hypophosphorylation of BicD, and genetic evidence indicates that this phosphorylation is crucial for BicD function. Polo overexpression might restore a functional level of BicDPA66 phosphorylation. Therefore, even if no significant changes were observed in the gel mobility of BicD in polo hypomorph mutants, it is tempting to propose that the function of Polo in the polarized transport could be to activate, directly or indirectly, BicD by phosphorylation (Mirouse, 2006).
Taken together, these results lead to a model that can explain a reciprocal requirement between the control of meiosis and oocyte specification. This model is based on four major points: (1) BicD is required for the Dynein-dependent polarized transport of oocyte determinants; (2) BicD is also required for the progressive localization of Polo to the oocyte; (3) Polo appears to trigger meiosis in the germarium; (4) Polo is required to activate the BicD and Dynein-dependent polarized transport. These findings suggest the existence of a positive feedback loop between Polo and BicD proteins, and therefore between oocyte specification and meiosis (Mirouse, 2006).
Two transcripts, one of 3.8 kb and the other of 4.4 kb are detected in RNA prepared from adult females. A third transcript of 5.7 kb is found in late embryos, pupae and adult males. The two transcripts differ in the length of the 3' tail. There are multiple polyadenylation signals that determine transcript length (Suter, 1989)
Bases in 5' UTR - 934
Bases in 3' UTR - 912
The sequence of Bic-D shows some similarity to the rod region of myosin heavy chains and to lamin, desmin, keratin, and other intermediate filament proteins, but there is no reason to believe that the protein is a myosin motor. The common feature of fibrous proteins such as the myosin heavy chain, is an extended alpha-helical coiled-coil structure that is built with a characteristic heptad repeat pattern, with hydrophobic residues at the first and fourth position (Suter, 1989 and Wharton, 1989).
A cDNA fragment homologous to the Drosophila Bicaudal-D gene (Bic-D) has been isolated using a hybridization selection procedure with cosmids derived from the short arm of human chromosome 12. A PCR-mediated cDNA cloning strategy was applied to obtain the coding sequence of the human homolog (BICD1) and to generate a partial mouse (Bicdh1) cDNA. The Drosophila Bicaudal-D gene encodes a coiled coil protein, characterized by five alpha-helix domains and a leucine zipper motif; the Drosophila protein forms part of the cytoskeleton and mediates the correct sorting of mRNAs for oocyte- and axis-determining factors during oogenesis. Analysis of the predicted amino acid sequence of the BICD1 cDNA clones indicates that the sequence similarity is essentially limited to the amphipatic helices and the leucine zipper, but the conserved order of these domains suggests a similar function of the protein in mammalians. A database search further indicates the existence of a second human homolog on chromosome arm 9q and a Caenorhabditis elegans homolog. Northern blot analysis indicates that both the human and the murine homologs are expressed in brain, heart, and skeletal muscle and during mouse embryonic development. The conserved structural characteristics of the BICD1 protein and its expression in muscle and especially brain suggest that BICD1 is a component of a cytoskeleton-based mRNA sorting mechanism conserved during evolution (Baens, 1997).
Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. A mammalian homolog of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein-dynactin interaction on the surface of membranous organelles, by associating with these complexes (Hoogenraad, 2001).
The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. A yeast two-hybrid approach has been used to identify binding partners of Rab6a. In particular, the dynein-dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologs of Drosophila Bicaudal-D, was identifed. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein-dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)-Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein-dynactin complex controls COPI-independent Golgi-ER transport (Matanis, 2002).
Human Nek8 is a new mammalian NIMA-related kinase, and its candidate substrate is Bicd2. Nek8 was isolated as a beta-casein kinase activity in rabbit lung and has an N-terminal catalytic domain homologous to the Nek family of protein kinases. Nek8 also contains a central domain with homology to RCC1, a guanine nucleotide exchange factor for the GTPase Ran, and a C-terminal coiled-coil domain. Like Nek2, Nek8 prefers beta-casein over other exogenous substrates, has shared biochemical requirements for kinase activity, and is capable of autophosphorylation and oligomerization. Nek8 activity is not cell cycle regulated, but like Nek3, levels are consistently higher in G(0)-arrested cells. During the purification of Nek8 a second protein co-chromatographed with Nek8 activity. This protein, Bicd2, is a human homolog of the Drosophila protein Bicaudal D, a coiled-coil protein. Bicd2 is phosphorylated by Nek8 in vitro, and the endogenous proteins associate in vivo. Bicd2 localizes to cytoskeletal structures, and its subcellular localization is dependent on microtubule morphology. Treatment of cells with nocodazole leads to dramatic reorganization of Bicd2, and correlates with Nek8 phosphorylation. This may be indicative of a role for Nek8 and Bicd2 associated with cell cycle independent microtubule dynamics (Holland, 2002).
Bicaudal D is an evolutionarily conserved protein that is involved in dynein-mediated motility both in Drosophila and in mammals. The N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. The BICD2 N-terminal domain has been used as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. These findings provide a novel model system for dissection of the molecular mechanism of dynein motility (Hoogenraad, 2003).
In mammals, two homologs of Bicaudal D, BICD1 and BICD2, are present (Baens, 1997; Hoogenraad, 2001). Studies in cultured mammalian cells have shown that BICD proteins bind to the small GTPase Rab6, as well as to dynein and dynactin complexes, and therefore participate in recruitment of dynein motor to Rab6-positive membranes of the Golgi apparatus and cytoplasmic vesicles (Hoogenraad, 2001; Matanis., 2002; Short, 2002). However, in addition to BICD proteins, Rab6 GTPase can also interact directly with the p150Glued component of the dynactin complex (Short et al., 2002). This raises the possibility that BICD acts as an accessory factor for the dynein motor, but is not sufficient by itself to recruit it to organelles (Hoogenraad, 2003 and references therein).
BICD proteins consist of several coiled-coil domains, and previous studies have demonstrated that while the C-terminal domain is responsible for interaction with membranes via Rab6, the N-terminal domain binds to cytoplasmic dynein (Hoogenraad, 2001; Matanis, 2002). In addition, the N- and C-terminal domains of BICD can interact with each other. Based on these findings, it is proposed that when BICD binds to the cargo (cytoplasmic vesicle) via its C-terminal domain, the N-terminal domain of BICD2 becomes available for interaction with dynein motor, which, in its turn, would transport the vesicle. If this model is correct, tethering of the BICD N-terminus to membranous organelles, which are normally devoid of BICD (such as mitochondria or peroxisomes), should be sufficient to induce their transport by cytoplasmic dynein. In this study, this idea is tested, and it is shown that the N-terminal part of BICD2 protein is indeed a potent recruitment factor for dynein, and that it can act in different molecular contexts (Hoogenraad, 2003).
Multiple approaches were used to investigate (1) the role of Rab6 relative to Zeste White 10 (ZW10), a mitotic checkpoint protein implicated in Golgi/endoplasmic reticulum (ER) trafficking/transport, and (2) conserved oligomeric Golgi (COG) complex, a putative tether in retrograde, intra-Golgi trafficking. ZW10 depletion resulted in a central, disconnected cluster of Golgi elements and inhibition of ERGIC53 and Golgi enzyme recycling to ER. Small interfering RNA (siRNA) against RINT-1, a protein linker between ZW10 and the ER soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 18, produced similar Golgi disruption. COG3 depletion fragmented the Golgi and produced vesicles; vesicle formation was unaffected by codepletion of ZW10 along with COG, suggesting ZW10 and COG act separately. Rab6 depletion did not significantly affect Golgi ribbon organization. Epistatic depletion of Rab6 inhibited the Golgi-disruptive effects of ZW10/RINT-1 siRNA or COG inactivation by siRNA or antibodies. Dominant-negative expression of guanosine diphosphate-Rab6 suppressed ZW10 knockdown induced-Golgi disruption. No cross-talk was observed between Rab6 and endosomal Rab5, and Rab6 depletion failed to suppress p115 (anterograde tether) knockdown-induced Golgi disruption. Dominant-negative expression of a C-terminal fragment of Bicaudal D, a linker between Rab6 and dynactin/dynein, suppressed ZW10, but not COG, knockdown-induced Golgi disruption. It is concluded that Rab6 regulates distinct Golgi trafficking pathways involving two separate protein complexes: ZW10/RINT-1 and COG (Sun, 2007).
The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, its intracellular environment and dynamic behavior were characterized. A Y-2H search for novel Rab6 interacting proteins identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. It is concluded that the brain-specific Rab6B is linked via Bicaudal-D1 to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells (Wanschers, 2007).
Chlamydia species are obligate intracellular bacteria that replicate within a membrane-bound vacuole, the inclusion, which is trafficked to the peri-Golgi region by processes that are dependent on early chlamydial gene expression. Although neither the host nor the chlamydial proteins that regulate the intracellular trafficking have been clearly defined, several enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, including Rab6, are recruited to Chlamydia trachomatis inclusions. To further characterize the association of Rab6 with C. trachomatis inclusions, the intracellular localization of guanine nucleotide-binding mutants of Rab6 was examined, and it was demonstrated that only active GTP-bound and not inactive GDP-bound EGFP-Rab6 mutants were recruited to the inclusion, suggesting that EGFP-Rab6 interacts with the inclusion via a host Rab6 effector or a chlamydial protein that mimics a Rab6 effector. Using EGFP-tagged fusion proteins, it was also demonstrated that the Rab6 effector Bicaudal D1 (BICD1) localized to C. trachomatis inclusions in a biovar-specific manner. In addition, EGFP-Rab6 and its effector EGFP-BICD1 are recruited to the inclusion in a microtubule- and Golgi apparatus-independent but chlamydial gene expression-dependent mechanism. Finally, in contrast to the Rab6-dependent Golgi apparatus localization of endogenous BICD1, EGFP-BICD1 was recruited to the inclusion by a Rab6-independent mechanism. Collectively, these data demonstrate that neither Rab6 nor BICD1 is trafficked to the inclusion via a Golgi apparatus-localized intermediate, suggesting that each protein is trafficked to the C. trachomatis serovar L2 inclusion by a unique, but as-yet-undefined, mechanism (Moorhead, 2007).
date revised: 1 March 2004
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