Rytis Prekeris

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

The Rip11/Rab11-FIP5 and kinesin II complex regulates endocytic protein recycling.

Sorting and recycling of endocytosed proteins are required for proper cellular function and growth. Internalized receptors either follow a fast constitutive recycling pathway, returning to the cell surface directly from the early endosomes, or a slow pathway that involves transport via perinuclear recycling endosomes. Slow recycling pathways are thought to play a key role in directing recycling proteins to specific locations on cell surfaces, such as the leading edges of motile cells. These pathways are regulated by various Rab GTPases, such as Rab4 and Rab11. Here we characterize the role of Rip11/FIP5, a known Rab11-binding protein, in regulating endocytic recycling. We use a combination of electron and fluorescent microscopy with siRNA-based protein knockdown to show that Rip11/FIP5 is present at the peripheral endosomes, where it regulates the sorting of internalized receptors to a slow recycling pathway. We also identify kinesin II as a Rip11/FIP5-binding protein and show that it is required for directing endocytosed proteins into the same recycling pathway. Thus, we propose that the Rip11/FIP5-kinesin-II complex has a key role in the routing of internalized receptors through the perinuclear recycling endosomes.

Transport at the recycling endosome.

The recycling endosome (RE) has long been considered as a sub-compartment of the early endosome that recycles internalized cargoes to the plasma membrane. The RE is now appreciated to participate in a more complex set of intracellular itineraries. Key cargo molecules and transport factors that act in these pathways are being identified. These advancements are beginning to reveal complexities in pathways involving the RE, and also suggest ways of further delineating functional domains of this compartment.

Breaking up is hard to do - membrane traffic in cytokinesis.

Throughout normal development, and in aberrant conditions such as cancer, cells divide by a process called cytokinesis. Most textbooks suggest that animal cells execute cytokinesis using an actomyosin-containing contractile ring, whereas plant cells generate a new cell wall by the assembly of a novel membrane compartment using vesicle-trafficking machinery in an apparently distinct manner. Recent studies have shown that cytokinesis in animal and plant cells may not be as distinct as these models imply – both have an absolute requirement for vesicle traffic. Moreover, some of the key molecular components of cytokinesis have been identified, many of which are proteins that function to control membrane traffic. Here, we review recent advances in this area.

Formation of Mutually Exclusive Rab11 Complexes with Members of the Family of Rab11-interacting Proteins Regulates Rab11 Endocytic Targeting and Function

Several Rabs, including Rab11, regulate the traffic and sorting of proteins in the endosomal pathway. Recently, six novel Rab11 family interacting proteins (FIPs) were identified. Although they share little overall sequence homology, all FIPs contain a conserved Rab11-binding domain. Here we investigate the role of FIPs as Rab11-targeting proteins and show that the Rab11-binding domain assumes an α-helical structure, with the conserved residues forming a hydrophobic Rab11-binding patch. This hydrophobic patch mediates the formation of mutually exclusive complexes between Rab11 and various members of FIP protein family. Furthermore, the formation of Rab11/FIP complexes regulates Rab11 localization by recruiting it to distinct endocytic compartments. Thus, we propose that Rab11/FIP complexes serve as targeting patches, regulating Rab11 localization and recruitment of additional cellular factors to different endocytic compartments.

FIP3-endosome-dependent formation of the secondary ingression mediates ESCRT-III recruitment during cytokinesis

The final cytokinesis event involves severing of the connecting intercellular bridge (ICB) between daughter cells. FIP3-positive recycling endosomes (FIP3 endosomes) and ESCRT complexes have been implicated in mediating the final stages of cytokinesis. Here we analyse the spatiotemporal dynamics of the actin cytoskeleton, FIP3-endosome fusion and ESCRT-III localization during cytokinesis to show that the ICB narrows by a FIP3-endosome-mediated secondary ingression, whereas the ESCRT-III complex is needed only for the last scission step of cytokinesis. We characterize the role of FIP3 endosomes during cytokinesis to demonstrate that FIP3 endosomes deliver SCAMP2/3 and p50RhoGAP to the ICB during late telophase, proteins required for the formation of the secondary ingression. We also show that the FIP3-endosome-induced secondary ingression is required for the recruitment of the ESCRT-III complex to the abscission site. Finally, we characterize a FIP3-endosome-dependent regulation of the ICB cortical actin network through the delivery of p50RhoGAP. These results provide a framework for the coordinated efforts of actin, FIP3 endosomes and the ESCRTs to regulate cytokinesis and abscission.

Endocytic membrane fusion and buckling-induced microtubule severing mediate cell abscission.

Cytokinesis and abscission are complicated events that involve changes in membrane transport and cytoskeleton organization. We have used the combination of time-lapse microscopy and correlative high-resolution 3D tomography to analyze the regulation and spatio-temporal remodeling of endosomes and microtubules during abscission. We show that abscission is driven by the formation of a secondary ingression within the intracellular bridge connecting two daughter cells. The initiation and expansion of this secondary ingression requires recycling endosome fusion with the furrow plasma membrane and nested central spindle microtubule severing. These changes in endosome fusion and microtubule reorganization result in increased intracellular bridge plasma membrane dynamics and abscission. Finally, we show that central spindle microtubule reorganization is driven by localized microtubule buckling and breaking, rather than by spastin-dependent severing. Our results provide a new mechanism for mediation and regulation of the abscission step of cytokinesis.

Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid‐body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk‐4 is a FIP3‐binding protein. Furthermore, we show that FIP3 binds to Cyk‐4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid‐body. We have mapped the FIP3‐binding region on Cyk‐4 and show that it overlaps with the ECT2‐binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk‐4 and that dissociation of ECT2 from the mid‐body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto‐myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.

The regulation of MMP targeting to invadopodia during cancer metastasis.

The dissemination of cancer cells from the primary tumor to a distant site, known as metastasis, is the main cause of mortality in cancer patients. Metastasis is a very complex cellular process that involves many steps, including the breaching of the basement membrane (BM) to allow the movement of cells through tissues. The BM breach occurs via highly regulated and localized remodeling of the extracellular matrix (ECM), which is mediated by formation of structures, known as invadopodia, and targeted secretion of matrix metalloproteinases (MMPs). Recently, invadopodia have emerged as key cellular structures that regulate the metastasis of many cancers. Furthermore, targeting of various cytoskeletal modulators and MMPs has been shown to play a major role in regulating invadopodia function. Here, we highlight recent findings regarding the regulation of protein targeting during invadopodia formation and function.

Arf GTPase-activating protein ASAP1 interacts with Rab11 effector FIP3 and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome.

ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment.