Nuf is a structural and functional homolog of Arfo2 (Hickson, 2003) and contains a highly conserved 20-aa Rab11-binding site. This binding domain was first identified by Prekeris (2001) and Hales (2001) as important for the interaction between Rab11 and a novel family of putative Rab11 effector proteins. Within this domain, Nuf and Arfo2 contain eight identical and six conserved amino acids. Nuf and hRip11, a mammalian Rab11 effector protein, contain ten identical and three conserved amino acids. This sequence conservation, combined with the colocalization results, prompted an examination of whether Nuf and Rab11 physically interact. Bacterially expressed GST-Rab11 was mixed with CHO cells transiently expressing GFP-Nuf. GTPgammaS and GDPßS were added to the buffer to test the nucleotide specificity of the interaction. GFP-Nuf is effectively pulled down by both GST-Rab11+GTPgammaS and GST-Rab11+GDPßS, indicating that the interaction is not tightly linked to the state of the nucleotide. GST-Rab11+GDPßS pulls down Nuf to a lesser extent than GST-Rab11+GTPgammaS. Nucleotide-independent binding has also been observed with other Rab11 effectors, Rab11-FIP2 (Hales, 2001) and Arfo2, the mammalian homolog of Nuf (Hickson, 2003). To test the specificity of the interaction, similar pull-down experiments were performed with Rab5, a component of the early endosome. Unlike the results with GST-Rab11, GFP-Nuf is not pulled down by GST-Rab5 in either the activated or unactivated form (Riggs, 2003).
To determine if Nuf is required for pericentriolar Rab11 localization, Rab11 (Rab-protein 11) localization was examined in nuf-derived embryos. Rab11 exhibits a concentrated punctate distribution around the centrosome during prophase. In nuf-derived embryos, both the punctate distribution and concentration of Rab11 around the centrosomes is completely abolished. Although levels of Nuf at the centrosome are greatly reduced during metaphase, Nuf is required for Rab11 centrosome localization at this stage as well. Nuf is also required for Rab11 localization during cellularization. The robust tight localization of Rab11 around the centrosome during cellularization is absent in nuf-derived embryos. It is believed that mislocalization of Rab11 in nuf is not a result of a general disruption of the intracellular transport pathway, since staining with Golgi marker Lava-lamp revealed normal Golgi distribution throughout the cell cycle in wild-type and nuf-derived embryos. From this analysis, it cannot be determined whether levels of Rab11 protein are reduced in nuf-derived embryos (Riggs, 2003).
Whether Rab11 is required for normal pericentriolar Nuf localization was also examined. Because Rab11 is an essential gene, a combination of hypomorphic rab11 alleles were used that permitted normal zygotic development (Jankovics, 2001). However, these transheterozygote females produced embryos with reduced levels of maternally supplied Rab11 and showed a reduced hatch rate. Wild-type and rab11-derived embryos were double stained for Nuf and DNA, and were examined during the syncytial divisions and cellularization. During prophase, while the pericentriolar localization of Nuf was robust in control embryos, pericentriolar Nuf levels were absent in rab11-derived embryos. The same result was obtained when cellularizing rab11-derived embryos were examined; the normal pericentriolar localization of Nuf is completely abolished. From this analysis, it cannot be determined whether levels of Nuf protein are reduced in rab11-derived embryos. These experiments demonstrate that Nuf and Rab11 are mutually dependent on one another for their localization to the RE (Riggs, 2003).
Drosophila sensory organ precursor (SOP) cells are a well-studied model
system for asymmetric cell division. During SOP division, the determinants Numb
and Neuralized segregate into the pIIb daughter cell and establish a distinct
cell fate by regulating Notch/Delta signaling. This study describes a Numb- and
Neuralized-independent mechanism that acts redundantly in cell-fate
specification. Trafficking of the Notch ligand Delta is different
in the two daughter cells. In pIIb, Delta passes through the recycling endosome
which is marked by Rab11. In pIIa, however, the recycling endosome does not form
because the centrosome fails to recruit Nuclear fallout, a Rab11 binding partner
that is essential for recycling endosome formation. Using a mammalian cell
culture system, it was demonstrated that recycling endosomes are essential for Delta
activity. These results suggest that cells can regulate signaling pathways and
influence their developmental fate by inhibiting the formation of individual
endocytic compartments (Emery, 2005).
To test whether Rab11 asymmetry is important for cell-fate specification, Rab11
accumulation in the pIIa cell was induced by nuf expression. Postorbital ES organs,
which can easily be scored in fairly high numbers, were used. Cell-fate transformations
upon nuf overexpression have been described (Abdelilah-Seyfried, 2000), but surprisingly, they do not
occur at high frequency. Such transformations can, however, be observed upon
coexpression of constitutively active Rab11. Upon expression of
nonphosphorylatable lgl, Numb and Neuralized asymmetry are disrupted, but
most ES organs still develop normally. When both pathways are disrupted by
coexpression of lgl3A and nuf, however, a large fraction of ES
organs shows cell-fate transformations that are consistent with a higher level
of Delta activity in pIIa. Lineage analysis shows cell-fate transformations in
44% of postorbital ES organs, and in 18% of these, pIIb cells are transformed
into pIIa cells (6% in ES organs expressing lgl3A alone). Twenty-five
percent of the cell fate transformations affect the first (SOP) while 75% affect
the second (pIIa) division, indicating that Rab11 asymmetry also plays a role in
other divisions of the SOP lineage. Taken together, these results suggest that two
partially redundant pathways exist to generate asymmetry in the SOP lineage: the
Par proteins phosphorylate Lgl to direct Numb and Neuralized into the pIIb cell
where they repress Notch or activate Delta, respectively. In the pIIa cell,
inhibition of Nuf and Rab11 inhibits Delta by preventing its trafficking through
the recycling endosome (Emery, 2005).
These results suggest that cells can also regulate signal
transduction pathways by controlling the formation or distribution of whole
endocytic compartments. After SOP division, Rab11-positive vesicles accumulate
around the centrosome in this cell but not in pIIa. Rab11 plays a
well-documented role in controlling vesicular protein transport through
recycling endosomes to the plasma membrane (Zerial, 2001). Dominant-negative forms of Rab11
inhibit the recycling of endocytosed Transferrin receptors or recruitment of
H+-K+-ATPase to the plasma membrane suggesting that Rab11
regulates trafficking of vesicular cargo through the recycling endosomal
compartment. In SOP cells, the asymmetric localization of Rab11 reflects a
different ability of pIIa and pIIb cells to recycle the Notch ligand Delta.
Rab11 asymmetry is observed 3.5 min after cytokinesis but Delta is in recycling
endosomes only 15 min after endocytosis. Thus, the protein is endocytosed before
mitosis and recycles back to the plasma membrane in pIIb but not in pIIa.
In pIIa, more Delta/Hrs double-positive
vesicles are observed, indicating that the protein enters a late-endosomal pathway (Emery, 2005).
Several observations indicate that
passage through recycling endosomes is essential for Delta to signal.
In a marrow stromal cell line, OP9,
inhibition of recycling endosomes dramatically reduces Delta signaling
capacity. Similarly, blocking the recycling pathway by overexpression of a
dominant-negative form of Rab11 in SOP cells causes relocalization of Delta into
enlarged late endosomes. In Drosophila wing discs, Delta has been
postulated to pass through a specific endocytic recycling pathway to acquire
signaling capacity (Wang,
2004). Finally, Jafar-Nejad (2005) demonstrates that the Rab11 binding
partner Sec15 is required both for Delta trafficking and Notch activation in the
SOP lineage. Sec15 is a component of the exocyst and is a Rab11
effector (Zhang, 2004). Although Sec15 is not asymmetric itself, it is conceivable that the higher
amounts of GTP bound Rab11 in pIIb increase its activity in delivering Delta to
the plasma membrane. A difference between Delta trafficking in pIIa and pIIb has
been observed previously (Le Borgne, 2003), but both Delta/Hrs vesicles and total number of Delta
vesicles were actually higher in pIIb in these previous experiments. While these earlier
experiments analyzed the whole two cell stage, this study focusses on the short time
interval right after mitosis where Rab11 is asymmetric. This explains the
different outcome and might in fact indicate that pIIb cells switch from an
initial phase where Delta is recycled to a later phase where trafficking is
regulated by neuralized-dependent endocytosis (Emery, 2005).
Although many cell types in different organisms undergo asymmetric cell division, only one
mechanism has been identified so far that directs this important biological
process in animals. This mechanism involves the Par proteins, which
phosphorylate Lgl on one side and direct cell fate determinants to the opposite
side of the cell cortex. Several results indicate that other pathways might
exist: in dividing progenitor cells of the mammalian brain, Numb segregates into
one of the two daughter cells and is required for lineage specification. However, some of
these divisions are asymmetric, although their orientation predicts that Numb
would be inherited by both daughter cells. In Drosophila SOP cells,
lgl3A overexpression affects both Numb and Neuralized localization
but has only a minor influence on the asymmetric outcome of the
division. The results indicate that the
asymmetric distribution of Rab11 is established through a distinct pathway:
(1) Rab11 asymmetry is unaffected in SOP cells overexpressing lgl3A;
(2) Rab11 is still asymmetric in dlg mutants where Par proteins do
not localize and Numb and Neuralized segregate into both daughter cells; (3)
Rab11 asymmetry can be uncoupled from Numb and Neuralized localization by the
expression of inscuteable; (4) the events responsible for Rab11
asymmetry seem to occur in the pIIa cell, but none of the known determinants is
inherited by this daughter cell. Although the observations could also be
explained if Numb or Neuralized would relieve a general suppression of recycling
endosome formation in the SOP lineage, this is unlikely since Rab11 asymmetry is
unaffected in numb or neuralized mutants. More likely, an unknown
factor could act on Nuf or the centrosome in the pIIa cell to prevent Rab11
accumulation. Nuf localization is cell cycle regulated, and a key regulatory
component could be missing in pIIa. For example, Nuf is highly phosphorylated and
differential activity of a kinase or phosphatase could prevent its
pericentriolar localization in the pIIa cell. Homologs of Nuf exist and bind to
Rab11 in vertebrates. Their expression pattern has not yet been described but it
will be interesting to determine whether these homologs regulate Notch signaling
in vertebrates and are responsible for asymmetric cell division in the mammalian
brain (Emery, 2005).
Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, this study demonstrates that microtubules are required for proper metaphase furrow formation. It is proposed that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows (Riggs, 2007; full text of article).
Microtubule-based motility has been implicated in many steps in endocytosis, and there is increasing evidence that it influences the distribution and activity of endocytic organelles. The work presented in this study suggests that motor-based movement of Rab effectors may be another means of regulating endosomal activity. Previous studies have shown that the Drosophila Rab11 effector, Nuf, is required for stable Rab11 localization at the RE and thus RE activity. Nuf concentrates at the MTOC during interphase through prophase and disperses into the cytoplasm at metaphase. This study demonstrates that Nuf relies on microtubules and minus-end microtubule motor Dynein both for its accumulation and maintenance at the MTOC. This raises the possibility that the Dynein-dependent delivery of Nuf to the RE may play a role in regulating Rab11 activity at the RE. Significantly maximal localization of Nuf at the MTOC-associated RE occurs during late interphase and prophase. This is the time of the establishment and formation of the metaphase furrows, which rely on RE-based vesicle delivery (Riggs, 2007).
Immunoprecipitation data demonstrates a physical interaction between Nuf and Dynein. This raises the possibility that the cell cycle-regulated localization of Nuf at the MTOC is mediated by a corresponding cell cycle-regulated interaction between Nuf and Dynein. Support for this idea comes from a study in vertebrate cells, demonstrating that Polo-like kinase (Plk) mediated phosphorylation of Ninein-like protein (Nlp), a microtubule-nucleating protein, directly determines its cell cycle-regulated localization at the centrosome. Like Nuf, Nlp localizes to the centrosome by associating with the minus-end-directed motor protein Dynein. As cells progress into metaphase, Plk is activated and phosphorylates Nlp on sites that are required for its association with Dynein. This disrupts Nlp ability to associate with Dynein and results in loss of Nlp from the centrosome (Riggs, 2007).
There is a strong correlation between maximal Nuf localization at the MTOC and furrow invagination. During the cortical divisions, furrow invagination and maximal Nuf concentration at the MTOC occurs during prophase. During cellularization, furrow invagination and maximal Nuf concentration at the MTOC occurs during interphase. Stable localization of Nuf and Rab11 at the MTOC during cellularization enabled a demonstration that microtubules are continuously required for maintaining Nuf and Rab11 at the MTOC. Colchicine-induced disruption of the interphase microtubules results in the rapid loss of Nuf from the MTOC. One interpretation of this result is that colchicine disrupts MTOC organization, which is required for maintaining Nuf at the MTOC. In contrast to the colchicine injections, injecting anti-Dynein antibody does not alter microtubule organization and results in a slow steady decrease of Nuf at the MTOC. This result suggests that the steady-state level of Nuf at the MTOC is maintained by continuous Dynein-dependent recruitment of Nuf to the MTOC. This also implies that Nuf is continuously released from the MTOC as well. The mechanism driving the release is unclear. Previous live analysis revealed vectorial movement of Nuf away from the centrosome, suggesting that it may rely on a kinesin, a plus-end-directed microtubule motor. If kinesin is involved, this implies that the balance between plus- and minus-end motor activities dictates whether Nuf is concentrated at the MTOC or dispersed in the cytoplasm. Recent work indicates that the positioning and activity of the early endosome is mediated through a balance of plus- and minus-end motor activities. In addition, investigations into cellular furrow elongation demonstrated that Lava lamp, a Golgi-associated protein, is complexed with Dynein and is responsible for Golgi-based movements necessary for latter half of furrow elongation (Riggs, 2007 and references therein).
The above studies demonstrate that microtubules are continuously required for proper Nuf localization at the MTOC. This raises the possibility that microtubule-based localization of Nuf at the MTOC is necessary for its association with the Rab11 and proper RE function. Because RE function is necessary for metaphase furrow formation, this predicts that microtubules are required for proper metaphase furrow formation. However previous studies did not observe defects in furrow formation when embryos were treated with microtubule inhibitors. It has been concluded that microtubules are dispensable for proper metaphase furrow formation in the early embryo. This issue was reexamined by injecting microtubule inhibitors at precise times throughout the cell cycle during the syncytial divisions. Because disrupting the microtubules at metaphase activates the spindle assembly checkpoint, the embryos were injected immediately after entry into anaphase. In these experiments, the nuclear cycle progressed normally but formation of the metaphase furrows were profoundly disrupted. Incorporation of GFP-tagged Moesin into the furrows that form at the next prophase completely fails. Thus these experiments define anaphase as a key time in which microtubules are required for recruiting actin to the furrows that form in the following prophase. The previous study failed to appreciate the role of microtubules in metaphase furrow formation because it was not possible to produce disruptions in the microtubule network at defined stages of the cell cycle (Riggs, 2007).
These studies also revealed that injecting colchicine at telophase produced no defects in actin recruitment. Similar injections at interphase through prophase also produced no defects in actin recruitment to the metaphase furrows. One interpretation of these results is that microtubules are specifically required during anaphase but not telophase or later for furrow formation in the next prophase. However it must be pointed the different classes of microtubules are differentially sensitive to microtubule inhibitors. Thus this differential sensitivity may contribute to the observed cell phase sensitivity of metaphase furrow formation to colchicine (Riggs, 2007).
That microtubules are required during anaphase for metaphase furrow formation in the following prophase is significant for a number of reasons. First, these studies support, although certainly do not prove, a model in which microtubule-based transport of Nuf to the MTOC is necessary for normal metaphase furrow formation. Second, anaphase/telophase is the point at which the metaphase furrows begin to regress. Thus the timing of furrow regression corresponds to the time at which microtubules are involved in establishing the next round of furrow formation. This indicates that the speed of the cortical divisions is not only achieved by an accelerated nuclear cycle but also by overlapping furrow regression with furrow formation. During anaphase, the replicated centrosomes possess robust astral arrays and the midbody has not yet fully formed. It is hypothesized that the plus ends of these overlapping arrays from neighboring centrosomes define the position of the metaphase furrow in the next cell cycle. This readily explains why furrows encompass the spindle and do not form at the midzone microtubules. Finally, although the furrows form at prophase, these studies identify anaphase as a critical time in which furrow is established. This also corresponds to the time at which microtubules are required during conventional furrow formation (Riggs, 2007).
Plasma membrane ingression during cytokinesis involves both actin remodeling and vesicle-mediated membrane addition. Vesicle-based membrane delivery from the recycling endosome (RE) has an essential but ill-defined involvement in cytokinesis. In the Drosophila early embryo, Nuclear fallout, a Rab11 effector which is essential for RE function, is required for F-actin and membrane integrity during furrow ingression. In nuf mutant embryos, an initial loss of F-actin at the furrow is followed by loss of the associated furrow membrane. Wild-type embryos treated with Latrunculin A or Rho inhibitor display similar defects. Drug- or Rho-GTP-induced increase of actin polymerization or genetically mediated decrease of actin depolymerization suppresses the nuf mutant F-actin and membrane defects. RhoGEF2 does not properly localize at the furrow in nuf mutant embryos, and RhoGEF2-Rho1 pathway components show strong specific genetic interactions with Nuf. A model is proposed in which RE-derived vesicles promote furrow integrity by regulating the rate of actin polymerization through the RhoGEF2-Rho1 pathway (Cao, 2008).
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