No abstract

An integral part of cell division is the separation of daughter cells via cytokinesis.There is now good evidence that the completion of cytokinesis requires coordinated membrane trafficking to deliver new membrane to the tip of the furrow and to complete the abscission. Here we have examined membrane traffic in cytokinesis and describe several novel observations. First, we show that Rab11-and FIP3-containing recycling endosomes accumulate near the cleavage furrow and are required for successful completion of cytokinesis. Second, we demonstrate that the Rab11-FIP3 protein complex is intimately involved in the delivery of endosomes to the cleavage furrow. Significantly, although FIP3 recruitment to endosomes is Rab11 dependent, we find that the targeting of FIP3 to the midbody is independent of Rab11. Third, we show that the Rab11-FIP3 complex is required for a late stage of cytokinesis, possibly abscission. Finally, we demonstrate that localization of FIP3 is subject to substantial spatial and temporal regulation. These data provide the first detailed analysis of recycling endosomes in cell division and provide a new model for membrane traffic to the furrow. We propose that the dynamic Rab11-FIP3 interaction controls the delivery, targeting, and fusion of recycling endosomes with furrow during late cytokinesis and abscission. INTRODUCTIONAn integral part of cell division is the physical separation of two daughter cells via a process known as cytokinesis (Scholey et al., 2003). At least two distinct processes are required for successful cytokinesis: formation and constriction of an acto-myosin contractile ring and the delivery of new membrane to the progressing cleavage furrow (O'Halloran, 2000;Scholey et al., 2003). Both of these steps are tightly controlled and crucial for cell abscission, the final separation of the two cells. Although the function of the acto-myosin ring in cell division is well understood, we are only beginning to understand the role of membrane transport during cytokinesis. Evidence suggests that insertion of new membrane at the apex of cleavage furrow is crucial for the successful completion of cellularization in Drosophila embryos (Rothwell et al., 1999;Zhang et al., 2000). Similar requirements for membrane transport and fusion were also observed in Xenopus laevis eggs (Byers and Armstrong, 1986;Bieliavsky et al., 1992).The plasma membrane of the cleavage furrow is distinct in its lipid and protein composition from the rest of the plasma membrane (Emoto et al., 1996;Umeda and Emoto, 1999;Emoto and Umeda, 2000). The unique composition of cleavage furrow plasma membrane may underscore its ability to be deformed during ingression, as a cell is pinched in two, as well as possibly generating the signals that regulate progression of cytokinesis. Thus, in addition to the delivery of the membrane to compensate for the expanding plasma membrane surface, membrane traffic during cytokinesis could also mediate the delivery of proteins that control the ingression of the cleavage furrow as well as cell-...

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimidesensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts (Ϸ25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A͞SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A͞nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A͞SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

The dual Rab11/Arf binding proteins, family of Rab11-interacting proteins FIP3 and FIP4 function in the delivery of recycling endosomes to the cleavage furrow and are, together with Rab11, essential for completion of abscission, the terminal step of cytokinesis. Here, we report that both FIP3 and FIP4 bind Arf6 in a nucleotide-dependent manner but exhibit differential affinities for Rab11 and Arf6. Both FIP3 and FIP4 can form ternary complexes with Rab11 and Arf6. Arf6 is localised to the furrow and midbody and we show that Arf6-GTP functions to localise FIP3 and FIP4 to midbodies during cytokinesis. Exo70p, a component of the Exocyst complex, also localises to the furrow of dividing cells and interacts with Arf6. We show that depletion of Exo70p leads to cytokinesis failure and an impairment of FIP3 and Rab11 localisation to the furrow and midbody. Moreover, Exo70p co-immunoprecipitates FIP3 and FIP4. Hence, we propose that FIP3 and FIP4 serve to couple Rab11-positive vesicle traffic from recycling endosomes to the cleavage furrow/midbody where they are tethered prior to fusion events via interactions with Arf6 and the Exocyst.