The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R-YFP and DsRed-clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of 'unroofed' cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.     

Cooperative regulation of endocytic vesicle transport by yeast Eps15-like protein Pan1p and epsins

Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.     

Scd5p and clathrin function are important for cortical actin organization,endocytosis, and localization of sla2p in yeast

SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast. SCD5 is essential, but an scd5-Delta338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth. Further studies here demonstrate that scd5-Delta338 affects receptor-mediated and fluid-phase endocytosis and normal actin organization. The scd5-Delta338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars. scd5-Delta338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis. Moreover, Scd5p colocalizes with cortical actin. Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin. Overexpression of SCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Delta338 with a clathrin mutation exacerbates the actin and endocytic phenotypes. Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R. Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function. Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells.     

Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin–Hip1R interaction is involved in the cytoskeletal organization of coated pits.     

Novel function of clathrin light chain in promoting endocytic vesicle formation

Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy chains (HC) bound by a light chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p. Here, using in vivo analysis in yeast, we provide novel insight into the role of this interaction. We find that overexpression of LC partially restores endocytosis to cells lacking clathrin HC. This suppression is dependent on the Sla2p binding region of LC. Using live cell imaging techniques to visualize endocytic vesicle formation, we find that the N-terminal Sla2p binding region of LC promotes the progression of arrested Sla2p patches that form in the absence of HC. We propose that LC binding to Sla2p positively regulates Sla2p for efficient endocytic vesicle formation.     

Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.     

Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin

The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin-binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc1-Δ19-76 suppressed the internalization defects of null mutations in three late endocytic factors: amphiphysins (rvs161 and rvs167) and verprolin (vrp1). In actin sedimentation assays, CLC binding to Sla2 inhibited Sla2 interaction with F-actin. Furthermore, clc1-Δ19-76 suppression of the rvs and vrp phenotypes required the Sla2 actin-binding talin-Hip1/R/Sla2 actin-tethering C-terminal homology domain, suggesting that clc1-Δ19-76 promotes internalization by prolonging actin engagement by Sla2. We propose that CLC directs endocytic progression by pruning the Sla2-actin attachments in the clathrin lattice, providing direction for membrane internalization.     

Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast

Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Delta376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis.     

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.     

Negative regulation of yeast Eps15-like Arp2/3 complex activator, Pan1p, by the Hip1R-related protein, Sla2p, during endocytosis

Control of actin assembly nucleated by the Arp2/3 complex plays a crucial role during budding yeast endocytosis. The yeast Eps15-related Arp2/3 complex activator, Pan1p, is essential for endocytic internalization and proper actin organization. Pan1p activity is negatively regulated by Prk1 kinase phosphorylation after endocytic internalization. Phosphorylated Pan1p is probably then dephosphorylated in the cytosol. Pan1p is recruited to endocytic sites approximately 25 s before initiation of actin polymerization, suggesting that its Arp2/3 complex activation activity is kept inactive during early stages of endocytosis by a yet-to-be-identified mechanism. However, how Pan1p is maintained in an inactive state is not clear. Using tandem affinity purification-tagged Pan1p, we identified End3p as a stoichiometric component of the Pan1p complex, and Sla2p, a yeast Hip1R-related protein, as a novel binding partner of Pan1p. Interestingly, Sla2p specifically inhibited Pan1p Arp2/3 complex activation activity in vitro. The coiled-coil region of Sla2p was important for Pan1p inhibition, and a pan1 partial loss-of-function mutant suppressed the temperature sensitivity, endocytic phenotypes, and actin phenotypes observed in sla2DeltaCC mutant cells that lack the coiled-coil region. Overall, our results establish that Sla2p's regulation of Pan1p plays an important role in controlling Pan1p-stimulated actin polymerization during endocytosis.     

Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.     

Coupling actin dynamics to the endocytic process in Saccharomyces cerevisiae

Summary. Endocytosis is an essential eukaryotic process that, in many systems, has been reported to require a functional actin cytoskeleton. The process of endocytosis is critical for controlling the protein–lipid composition of the plasma membrane and uptake of nutrients as well as pathogens and also plays an important role in regulation of cell signalling. While several distinct pathways for endocytosis have been characterised, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly supported by studies in mammalian cells. Current evidence suggests that cortical patches are sites of endocytosis in Saccharomyces cerevisiae and that these sites are composed of sequentially forming protein complexes. Distinct stages in complex formation are characterised by the presence of different activators of F-actin polymerisation. Disassembly of the complexes is also essential for the endocytosis to proceed. Mutants lacking the kinases Ark1 and Prk1 accumulate actin and endocytic machinery in a single large clump in cells. Phosphorylation of endocytic proteins including Sla1p is proposed to cause their removal from the complex and allow later stages of the invagination process to occur. Dephosphorylation of endocytic components may then allow subsequent reincorporation into new sites of endocytic complex assembly. Correspondence and reprints: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Molecular requirements for the internalisation step of endocytosis: insights from yeast

Molecular genetic studies of endocytosis using the unicellular eukaryote Saccharomyces cerevisiae (budding yeast) have led to the identification of many cellular components, both proteins and lipids, required for this process. While initially, many of these requirements (e.g. for actin, various actin-associated proteins, the ubiquitin conjugation system, and for ergosterol and sphingolipids) appeared to differ from known requirements for endocytosis in higher eukaryotes (e.g. clathrin, AP-2, dynamin), it now seems that endocytosis in higher and lower eukaryotes share many requirements. Often, what were initially identified as actin cytoskeleton-associated proteins in S. cerevisiae, are now revealing themselves as clathrin-coated pit- and vesicle-associated proteins in higher eukaryotes. So rather than delineating two endocytic pathways, one actin-based and one clathrin-based, the combined studies on higher and lower eukaryotes are revealing interesting interplay in both systems between the actin cytoskeleton, clathrin coats, and lipids in the formation of endocytic vesicles at the plasma membrane. Recent results from the yeast system show that the Arp2/3p complex, Wiskott-Aldrich syndrome protein (WASP), and WASP-interacting protein (WIP), proteins involved in the nucleation step of actin filament assembly, play a major role in the formation of endocytic vesicles. This discovery suggests models whereby endocytic vesicles may be actively pushed from the plasma membrane and into the cell by newly forming and rapidly extending actin filaments.     

Structural definition of the F-actin-binding THATCH domain from HIP1R

Huntingtin-interacting protein-1 related (HIP1R) has a crucial protein-trafficking role, mediating associations between actin and clathrin-coated structures at the plasma membrane and trans-Golgi network. Here, we characterize the F-actin-binding region of HIP1R, termed the talin-HIP1/R/Sla2p actin-tethering C-terminal homology (THATCH) domain. The 1.9-A crystal structure of the human HIP1R THATCH core reveals a large sequence-conserved surface patch created primarily by residues from the third and fourth helices of a unique five-helix bundle. Point mutations of seven contiguous patch residues produced significant decreases in F-actin binding. We also show that THATCH domains have a conserved C-terminal latch capable of oligomerizing the core, thereby modulating F-actin engagement. Collectively, these results establish a framework for investigating the links between endocytosis and actin dynamics mediated by THATCH domain-containing proteins.     

The Sla2p talin domain plays a role in endocytosis in Saccharomyces cerevisiae

Clathrin-binding adaptors play critical roles for endocytosis in multicellular organisms, but their roles in budding yeast have remained unclear. To address this question, we created a quadruple mutant yeast strain lacking the genes encoding the candidate clathrin adaptors Yap1801p, Yap1802p, and Ent2p and containing a truncated version of Ent1p, Ent1DeltaCBMp, missing its clathrin-binding motif. This strain was viable and competent for endocytosis, suggesting the existence of other redundant adaptor-like factors. To identify these factors, we mutagenized the quadruple clathrin adaptor mutant strain and selected cells that were viable in the presence of full-length Ent1p, but inviable with only Ent1DeltaCBMp; these strains were named Rcb (requires clathrin binding). One mutant strain, rcb432, contained a mutation in SLA2 that resulted in lower levels of a truncated protein lacking the F-actin binding talin homology domain. Analyses of this sla2 mutant showed that the talin homology domain is required for endocytosis at elevated temperature, that SLA2 exhibits genetic interactions with both ENT1 and ENT2, and that the clathrin adaptors and Sla2p together regulate the actin cytoskeleton and revealed conditions under which Yap1801p and Yap1802p contribute to viability. Together, our data support the view that Sla2p is an adaptor that links actin to clathrin and endocytosis.     

The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1^AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1^AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1^AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.      

Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis.

The mammalian protein epsin is required for endocytosis. In this study, we have characterized two homologous yeast proteins, Ent1p and Ent2p, which are similar to mammalian epsin. An essential function for the highly conserved N-terminal epsin N-terminal homology (ENTH) domain was revealed using deletions and randomly generated temperature-sensitive ent1 alleles. Changes in conserved ENTH domain residues in ent1(ts) cells revealed defects in endocytosis and actin cytoskeleton structure. The Ent1 protein was localized to peripheral and internal punctate structures, and biochemical fractionation studies found the protein associated with a large, Triton X-100-insoluble pellet. Finally, an Ent1p clathrin-binding domain was mapped to the final eight amino acids (RGYTLIDL*) in the Ent1 protein sequence. Based on these and other data, we propose that the yeast epsin-like proteins are essential components of an endocytic complex that may act at multiple stages in the endocytic pathway.     

Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis

The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.     

Depolymerization of cortical actin inhibits UT-A1 urea transporter endocytosis but promotes forskolin-stimulated membrane trafficking

The cytoskeleton participates in many aspects of transporter protein regulation. In this study, by using yeast two-hybrid screening, we identified the cytoskeletal protein actin as a binding partner with the UT-A1 urea transporter. This suggests that actin plays a role in regulating UT-A1 activity. Actin specifically binds to the carboxyl terminus of UT-A1. A serial mutation study shows that actin binding to UT-A1's carboxyl terminus was abolished when serine 918 was mutated to alanine. In polarized UT-A1-MDCK cells, cortical filamentous (F) actin colocalizes with UT-A1 at the apical membrane and the subapical cytoplasm. In the cell surface, both actin and UT-A1 are distributed in the lipid raft microdomains. Disruption of the F-actin cytoskeleton by latrunculin B resulted in UT-A1 accumulation in the cell membrane as measured by biotinylation. This effect was mainly due to inhibition of UT-A1 endocytosis in both clathrin and caveolin-mediated endocytic pathways. In contrast, actin depolymerization facilitated forskolin-stimulated UT-A1 trafficking to the cell surface. Functionally, depolymerization of actin by latrunculin B significantly increased UT-A1 urea transport activity in an oocyte expression system. Our study shows that cortical F-actin not only serves as a structural protein, but directly interacts with UT-A1 and plays an important role in controlling UT-A1 cell surface expression by affecting both endocytosis and trafficking, therefore regulating UT-A1 bioactivity.