The functionally distinct fission yeast formins have specific actin-assembly properties

Fission yeast expresses three formins required for distinct actin cytoskeletal processes: Cdc12 (cytokinesis), For3 (polarization), and Fus1 (mating). We propose that in addition to differential regulation, key actin-assembly properties tailor formins for a particular role. In direct comparison to the well-studied Cdc12, we report the first in vitro characterization of the actin-assembly properties of For3 and Fus1. All three share fundamental formin activities; however, particular reaction rates vary significantly. Cdc12 is an efficient nucleator (one filament per approximately 3 Cdc12 dimers) that processively elongates profilin-actin at a moderate rate of 10 subunits s(-1) μM(-1), but lacks filament-bundling activity. Fus1 is also an efficient nucleator, yet processively elongates profilin-actin at one-half the rate of and dissociates 10-fold more rapidly than Cdc12; it also bundles filaments. For3 nucleates filaments 100-fold less well than Fus1, but like Cdc12, processively elongates profilin-actin at a moderate rate and lacks filament-bundling activity. Additionally, both the formin homology FH1 and FH2 domains contribute to the overall rate of profilin-actin elongation. We also confirmed the physiological importance of the actin-assembly activity of the fission yeast formins. Point mutants that disrupt their ability to stimulate actin assembly in vitro do not function properly in vivo.     

Effect of the pheromone-responsive G(alpha) and phosphatase proteins of Saccharomyces cerevisiae on the subcellular localization of the Fus3 mitogen-activated protein kinase

The mating-specific G(alpha) protein of Saccharomyces cerevisiae, Gpa1, stimulates adaptation to pheromone by a mechanism independent of G(beta gamma) sequestration. Genetic evidence suggests that Gpa1 targets the Fus3 mitogen-activated protein kinase, and it has recently been shown that the two proteins interact in cells responding to pheromone. To test the possibility that Gpa1 downregulates the mating signal by affecting the localization of Fus3, we created a Fus3-green fluorescent protein (GFP) fusion protein. In vegetative cells, Fus3-GFP was found in both the cytoplasm and the nucleus. Pheromone stimulated a measurable increase in the ratio of nuclear to cytoplasmic Fus3-GFP. In contrast, the relative level of nuclear Fus3-GFP decreased as cells recovered from pheromone arrest and did not increase when cells adapted to chronic stimulus were challenged again. Accumulation of Fus3-GFP in the nuclei of stimulated cells was also inhibited by overexpression of either wild-type Gpa1, the E364K hyperadaptive mutant form of Gpa1, or the Msg5 dually specific phosphatase. The effects of Gpa1 and Msg5 on Fus3 are partially interdependent. In a genetic screen for adaptive defective mutants, a nonsense allele of the nucleocytoplasmic transport receptor, Kap104, was identified. Truncation of the Kap104 cargo-binding domain blocked the effect of both Gpa1(E364K) and Msg5 on Fus3-GFP localization. Based on these results, we propose that Gpa1 and Msg5 work in concert to downregulate the mating signal and that they do so by inhibiting the pheromone-induced increase of Fus3 in the nucleus. Kap104 is required for the G(alpha)/phosphatase-mediated effect on Fus3 localization.     

Csi1 links centromeres to the nuclear envelope for centromere clustering

In the fission yeast Schizosaccharomyces pombe, the centromeres of each chromosome are clustered together and attached to the nuclear envelope near the site of the spindle pole body during interphase. The mechanism and functional importance of this arrangement of chromosomes are poorly understood. In this paper, we identified a novel nuclear protein, Csi1, that localized to the site of centromere attachment and interacted with both the inner nuclear envelope SUN domain protein Sad1 and centromeres. Both Csi1 and Sad1 mutants exhibited centromere clustering defects in a high percentage of cells. Csi1 mutants also displayed a high rate of chromosome loss during mitosis, significant mitotic delays, and sensitivity to perturbations in microtubule–kinetochore interactions and chromosome numbers. These studies thus define a molecular link between the centromere and nuclear envelope that is responsible for centromere clustering.     

Formin differentially utilizes profilin isoforms to rapidly assemble actin filaments

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.     

Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin: INSIGHTS FROM CRYSTAL STRUCTURES OF FISSION YEAST PROFILIN*S?

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161–1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider “wings” that interact with actin.The small protein profilin not only helps to maintain a cytoplasmic pool of actin monomers ready to elongate actin filament barbed ends (2), but it also binds to type II poly-l-proline helices (3, 4). The actin (5) and poly-l-proline (6–8) binding sites are on opposite sides of the profilin molecule, so profilin can link actin to proline-rich targets. Viability of fission yeast depends independently on profilin binding to both actin and poly-l-proline, although cells survive >10-fold reductions in affinity for either ligand (1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p (9, 10) and profilin (11) to assemble actin filaments for the cytokinetic contractile ring. Formins are multidomain proteins that nucleate and assemble unbranched actin filaments (12). Formin FH2 domains form homodimers that can associate processively with the barbed ends of growing actin filaments (13, 14). FH2 dimers slow the elongation of barbed ends (15). Most formin proteins have an FH1 domain linked to the FH2 domain. Binding profilin-actin to multiple polyproline sites in an FH1 domain concentrates actin near the barbed end of an actin filament associated with a formin FH2 homodimer. Actin transfers very rapidly from the FH1 domains onto the filament end (16) allowing profilin to stimulate elongation of the filament (15, 17).We tested the ability of human (Homo sapiens, Hs)⁷ profilin-I to complement the temperature-sensitive cdc3-124 mutation (11) in the single fission yeast profilin gene with the aim of using yeast to characterize human profilin mutations. The failure of expression of Hs profilin-I to complement the cdc3-124 mutation prompted us to compare human and fission yeast profilins more carefully. We report here a surprising incompatibility of Hs profilin-I with fission yeast formin Cdc12p, a crystal structure of fission yeast profilin, which allowed a detailed comparison with Hs profilin, and mutations that revealed how overexpression of Hs profilin-I compromises the viability of wild-type fission yeast.     

The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12–Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.     

Redistribution of centrosomal proteins by centromeres and Polo kinase controls partial nuclear envelope breakdown in fission yeast

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB—yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy, we show that the conserved Sad1-UNC-84 homology-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.     

Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly

During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.     

The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network.     

Formin-1 protein associates with microtubules through a peptide domain encoded by exon-2

Formin family proteins coordinate actin filaments and microtubules. The mechanisms by which formins bind and regulate the actin cytoskeleton have recently been well defined. However, the molecular mechanism by which formins coordinate actin filaments and microtubules remains poorly understood. We demonstrate here that Isoform-Ib of the Formin-1 protein (Fmn1-Ib) binds to microtubules via a protein domain that is physically separated from the known actin-binding domains. When expressed at low levels in NIH3T3 fibroblasts, Fmn1-Ib protein localizes to cytoplasmic filaments that nocodazole disruption confirmed as interphase microtubules. A series of progressive mutants of Fmn1-Ib demonstrated that deletion of exon-2 caused dissociation from microtubules and a stronger association with actin membrane ruffles. The exon-2-encoded peptide binds purified tubulin in vitro and is also sufficient to localize GFP to microtubules. Exon-2 does not contain any known formin homology domains. Deletion of exon 5, 7, 8, the FH1 domain or FH2 domain did not affect microtubule binding. Thus, our results indicate that exon-2 of Fmn1-Ib encodes a novel microtubule-binding peptide. Since formin proteins associate with actin filaments through the FH1 and FH2 domains, binding to interphase microtubules through this exon-2-encoded domain provides a novel mechanism by which Fmn1-Ib could coordinate actin filaments and microtubules.     

A Guaninine Nucleotide Exchange Factor Is a Component of the Meiotic Spindle Pole Body in Schizosaccharomyces pombe

Spore morphogenesis in yeast is driven by the formation of membrane compartments that initiate growth at the spindle poles during meiosis II and grow to encapsulate daughter nuclei. Vesicle docking complexes, called meiosis II outer plaques (MOPs), form on each meiosis II spindle pole body (SPB) and serve as sites of membrane nucleation. How the MOP stimulates membrane assembly is not known. Here, we report that SpSpo13, a component of the MOP in Schizosaccharomyces pombe, shares homology with the guanine nucleotide exchange factor (GEF) domain of the Saccharomyces cerevisiae Sec2 protein. ScSec2 acts as a GEF for the small Rab GTPase ScSec4, which regulates vesicle trafficking from the late-Golgi to the plasma membrane. A chimeric protein in which the ScSec2-GEF domain is replaced with SpSpo13 is capable of supporting the growth of a sec2Δ mutant. SpSpo13 binds preferentially to the nucleotide-free form of ScSec4 and facilitates nucleotide exchange in vitro. In vivo, a Spspo13 mutant defective in GEF activity fails to support membrane assembly. In vitro specificity experiments suggest that SpYpt2 is the physiological substrate of SpSpo13. These results demonstrate that stimulation of Rab-GTPase activity is a property of the S. pombe MOP essential for the initiation of membrane formation.     

Expression of multiple formins in adult tissues and during developmental stages of mouse brain

Formins are highly conserved heterogeneous family of proteins with several isoforms having significant contribution in multiple cellular functions. Formins play crucial role in remodelling of actin cytoskeleton and thus play important role in cell motility. Formins are also involved in many cellular activities like determining cell polarity, cytokinesis and morphogenesis. Formins are multi domain protein with characteristic homodimeric formin homology 2 (FH2) domain. It nucleates the actin filaments and its activity is regulated by the presence of characteristic formin homology 1 (FH1) domain. In higher mammals like human and mouse fifteen different formin isoforms are present. However the function and expression pattern of each and every formin in different adult tissues are not well characterized. Here we have found that multiple formins are expressing in each adult tissue of mouse, irrespective of their origin from the germ layer. Formins are also expressing from early stage of development to the adulthood in brain. The expression of many formins in a single tissue of adult mouse indicates that regulation of actin cytoskeleton dynamics by formins may be crucial for physiological processes like wound healing, tissue repairing, exocytosis, endocytosis, synapse formation and maintenance. Expression of FMNL2 and Fhdc1 are high in adult mouse brain as compare to embryonic stages. Higher expression of FMNL2 and Fhdc1 indicates that FMNL2 and Fhdc1 might be very important for the adult brain functions.     

Saccharomyces cerevisiae Kelch Proteins and Bud14 Protein Form a Stable 520-kDa Formin Regulatory Complex That Controls Actin Cable Assembly and Cell Morphogenesis

Formins perform essential roles in actin assembly and organization in vivo, but they also require tight regulation of their activities to produce properly functioning actin structures. Saccharomyces cerevisiae Bud14 is one member of an emerging class of formin regulators that target the FH2 domain to inhibit actin polymerization, but little is known about how these regulators are themselves controlled in vivo. Kelch proteins are critical for cell polarity and morphogenesis in a wide range of organisms, but their mechanistic roles in these processes are still largely undefined. Here, we report that S. cerevisiae Kelch proteins, Kel1 and Kel2, associate with Bud14 in cell extracts to form a stable 520-kDa complex with an apparent stoichiometry of 2:2:1 Bud14/Kel1/Kel2. Using pairwise combinations of GFP- and red fluorescent protein-tagged proteins, we show that Kel1, Kel2, and Bud14 interdependently co-localize at polarity sites. By analyzing single, double, and triple mutants, we show that Kel1 and Kel2 function in the same pathway as Bud14 in regulating Bnr1-mediated actin cable formation. Loss of any component of the complex results in long, bent, and hyper-stable actin cables, accompanied by defects in secretory vesicle traffic during polarized growth and septum formation during cytokinesis. These observations directly link S. cerevisiae Kelch proteins to the control of formin activity, and together with previous observations made for S. pombe homologues tea1p and tea3p, they have broad implications for understanding Kelch function in other systems.     

Arabidopsis Formin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785–amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes'' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes.     

RICE MORPHOLOGY DETERMINANT encodes the type II formin FH5 and regulates rice morphogenesis

Multicellular organisms contain a large number of formins; however, their physiological roles in plants remain poorly understood. Here, we reveal that formin homology 5 (FH5), a type II formin mutated in rice morphology determinant (rmd), plays a crucial role in determining rice (Oryza sativa) morphology. FH5/RMD encodes a formin-like protein consisting of an N-terminal phosphatase tensin (PTEN)-like domain, an FH1 domain, and an FH2 domain. The rmd mutants display a bending growth pattern in seedlings, are stunted as adult plants, and have aberrant inflorescence (panicle) and seed shape. Cytological analysis showed that rmd mutants have severe cell elongation defects and abnormal microtubule and microfilament arrays. FH5/RMD is ubiquitously expressed in rice tissues, and its protein localization to the chloroplast surface is mediated by the PTEN domain. Biochemical assays demonstrated that recombinant FH5 protein can nucleate actin polymerization from monomeric G-actin or actin/profilin complexes, cap the barbed end of actin filaments, and bundle actin filaments in vitro. Moreover, FH5 can directly bind to and bundle microtubules through its FH2 domain in vitro. Our findings suggest that the rice formin protein FH5 plays a critical role in determining plant morphology by regulating actin dynamics and proper spatial organization of microtubules and microfilaments.     

KAR5 Encodes a Novel Pheromone-inducible Protein Required for Homotypic Nuclear Fusion

KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.     

A role for nuclear envelope–bridging complexes in homology-directed repair

Unless efficiently and faithfully repaired, DNA double-strand breaks (DSBs) cause genome instability. We implicate a Schizosaccharomyces pombe nuclear envelope–spanning linker of nucleoskeleton and cytoskeleton (LINC) complex, composed of the Sad1/Unc84 protein Sad1 and Klarsicht/Anc1/SYNE1 homology protein Kms1, in the repair of DSBs. An induced DSB associates with Sad1 and Kms1 in S/G2 phases of the cell cycle, connecting the DSB to cytoplasmic microtubules. DSB resection to generate single-stranded DNA and the ATR kinase drive the formation of Sad1 foci in response to DNA damage. Depolymerization of microtubules or loss of Kms1 leads to an increase in the number and size of DSB-induced Sad1 foci. Further, Kms1 and the cytoplasmic microtubule regulator Mto1 promote the repair of an induced DSB by gene conversion, a type of homology-directed repair. kms1 genetically interacts with a number of genes involved in homology-directed repair; these same gene products appear to attenuate the formation or promote resolution of DSB-induced Sad1 foci. We suggest that the connection of DSBs with the cytoskeleton through the LINC complex may serve as an input to repair mechanism choice and efficiency.     

FRL, a novel formin-related protein, binds to Rac and regulates cell motility and survival of macrophages

We have isolated a cDNA, frl (formin-related gene in leukocytes), a novel mammalian member of the formin gene family. The frl cDNA encodes a 160-kDa protein, FRL, that possesses FH1, FH2, and FH3 domains that are well conserved among other Formin-related proteins. An FRL protein is mainly localized in the cytosol and is highly expressed in spleen, lymph node, and bone marrow cells. Formin-related genes and proteins have been reported to play crucial roles in morphogenesis, cell polarity, and cytokinesis through interaction with Rho family small GTPases. FRL binds to Rac at its N-terminal region including the FH3 domain and associates with profilin at the FH1 domain. In a macrophage cell line, P388D1, overexpression of a truncated form of FRL containing only the FH3 domain (FH3-FRL) strongly inhibited cell adhesion to fibronectin and migration upon stimulation with a chemokine. Moreover, expression of the truncated FH3-FRL protein resulted in apoptotic cell death of P388D1 cells, suggesting that the truncated FH3-FRL protein may interfere with signals of FRL. Overexpression in the P388D1 cells of full-length FRL or of the truncated protein containing the FH3 and FH1 domains, with simultaneous expression of the truncated FH3-FRL protein, blocked apoptotic cell death and inhibition of cell adhesion and migration. These results suggest that FRL may play a role in the control of reorganization of the actin cytoskeleton in association with Rac and also in the regulation of the signal for cell survival.     

PTRN‐1/CAMSAP promotes CYK‐1/formin‐dependent actin polymerization during endocytic recycling

Cargo sorting and membrane carrier initiation in recycling endosomes require appropriately coordinated actin dynamics. However, the mechanism underlying the regulation of actin organization during recycling transport remains elusive. Here we report that the loss of PTRN‐1/CAMSAP stalled actin exchange and diminished the cytosolic actin structures. Furthermore, we found that PTRN‐1 is required for the recycling of clathrin‐independent cargo hTAC‐GFP. The N‐terminal calponin homology (CH) domain and central coiled‐coils (CC) region of PTRN‐1 can synergistically sustain the flow of hTAC‐GFP. We identified CYK‐1/formin as a binding partner of PTRN‐1. The N‐terminal GTPase‐binding domain (GBD) of CYK‐1 serves as the binding interface for the PTRN‐1 CH domain. The presence of the PTRN‐1 CH domain promoted CYK‐1‐mediated actin polymerization, which suggests that the PTRN‐1‐CH:CYK‐1‐GBD interaction efficiently relieves autoinhibitory interactions within CYK‐1. As expected, the overexpression of the CYK‐1 formin homology domain 2 (FH2) substantially restored actin structures and partially suppressed the hTAC‐GFP overaccumulation phenotype in ptrn‐1 mutants. We conclude that the PTRN‐1 CH domain is required to stimulate CYK‐1 to facilitate actin dynamics during endocytic recycling.