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Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process., (© 2024. The Author(s).)

Oropouche fever, a debilitating illness common in South America, is caused by Oropouche virus (OROV), an arbovirus. OROV belongs to the Peribunyaviridae family, a large group of RNA viruses. Little is known about the biology of Peribunyaviridae in host cells, especially assembly and egress processes. Our research reveals that the small GTPase Rab27a mediates intracellular transport of OROV induced compartments and viral release from infected cells. We show that Rab27a interacts with OROV glycoproteins and colocalizes with OROV during late phases of the infection cycle. Moreover, Rab27a activity is required for OROV trafficking to the cell periphery and efficient release of infectious particles. Consistently, depleting Rab27a's downstream effector, Myosin Va, or inhibiting actin polymerization also hinders OROV compartments targeting to the cell periphery and infectious viral particle egress. These data indicate that OROV hijacks Rab27a activity for intracellular transport and cell externalization. Understanding these crucial mechanisms of OROV's replication cycle may offer potential targets for therapeutic interventions and aid in controlling the spread of Oropouche fever., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Concha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

N-methyl-D-aspartate receptor (NMDAR) trafficking is a key process in the regulation of synaptic efficacy and brain function. However, the molecular mechanism underlying the surface transport of NMDARs is largely unknown. Here we identified myosin Va (MyoVa) as the specific motor protein that traffics NMDARs in hippocampal neurons. We found that MyoVa associates with NMDARs through its cargo binding domain. This association was increased during NMDAR surface transport. Knockdown of MyoVa suppressed NMDAR transport. We further demonstrated that Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) regulates NMDAR transport through its direct interaction with MyoVa. Furthermore, MyoVa employed Rab11 family-interacting protein 3 (Rab11/FIP3) as the adaptor proteins to couple themselves with NMDARs during their transport. Accordingly, the knockdown of FIP3 impairs hippocampal memory. Together, we conclude that in hippocampal neurons, MyoVa conducts active transport of NMDARs in a CaMKII-dependent manner., (© 2024. The Author(s).)

Migrasomes, enriched with signaling molecules such as chemokines, cytokines and angiogenic factors, play a pivotal role in the spatially defined delivery of these molecules, influencing critical physiological processes including organ morphogenesis and angiogenesis. The mechanism governing the accumulation of signaling molecules in migrasomes has been elusive. In this study, we show that secretory proteins, including signaling proteins, are transported into migrasomes by secretory carriers via both the constitutive and regulated secretion pathways. During cell migration, a substantial portion of these carriers is redirected to the rear of the cell and actively transported into migrasomes, driven by the actin-dependent motor protein Myosin-5a. Once at the migrasomes, these carriers fuse with the migrasome membrane through SNARE-mediated mechanisms. Inhibiting migrasome formation significantly reduces secretion, suggesting migrasomes as a principal secretion route in migrating cells. Our findings reveal a specialized, highly localized secretion paradigm in migrating cells, conceptually paralleling the targeted neurotransmitter release observed in neuronal systems., (© 2024. The Author(s).)

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism., Competing Interests: Competing interests statement:The authors declare no competing interest.

An important question in cell biology is how cytoskeletal proteins evolved and drove the development of novel structures and functions. Here we address the origin of SPIRE actin nucleators. Mammalian SPIREs work with RAB GTPases, formin (FMN)-subgroup actin assembly proteins and class-5 myosin (MYO5) motors to transport organelles along actin filaments towards the cell membrane. However, the origin and extent of functional conservation of SPIRE among species is unknown. Our sequence searches show that SPIRE exist throughout holozoans (animals and their closest single-celled relatives), but not other eukaryotes. SPIRE from unicellular holozoans (choanoflagellate), interacts with RAB, FMN and MYO5 proteins, nucleates actin filaments and complements mammalian SPIRE function in organelle transport. Meanwhile SPIRE and MYO5 proteins colocalise to organelles in Salpingoeca rosetta choanoflagellates. Based on these observations we propose that SPIRE originated in unicellular ancestors of animals providing an actin-myosin driven exocytic transport mechanism that may have contributed to the evolution of complex multicellular animals., (© 2024. The Author(s).)

Mucormycosis is an emerging and deadly invasive fungal infection caused by fungi belonging to the Mucorales order. We investigated the myosin superfamily, which encompasses diverse actin-based motor proteins with various cellular functions. Specifically, the role of the Myo5B (ID 179665) protein from the myosin class V family in Mucor lusitanicus was explored by generating silencing phenotypes and null mutants corresponding to the myo5B gene. Silencing fungal transformants exhibited a markedly reduced growth rate and a nearly complete absence of sporulation compared to the wild-type strain. The myo5BΔ null mutant strain displayed atypical characteristics, including abnormally short septa and inflated hyphae. Notably, there were a majority of small yeast-like cells instead of filamentous hyphae in the mutant. These yeast-like cells cannot germinate normally, resulting in a loss of polarity. In vivo virulence assays conducted in the Galleria mellonella invertebrate model revealed that the myo5BΔ mutant strain was avirulent. These findings shed light on the crucial contributions of the Myo5B protein to the dimorphism and pathogenicity of M. lusitanicus . Therefore, the myosin V family is a potential target for future therapeutic interventions aimed at treating mucormycosis.

Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, that is, the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes., Competing Interests: JP, RZ, LY, JZ, NZ, QC, SS, XL No competing interests declared, (© 2024, Pan et al.)

Myosin Va (Myo Va) is one of three protein complexes involved in melanosome transport. In this study, we identified BMP-2 as an up-regulator of Myo Va expression using 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO). Our results showed that MNQO reduced the mRNA and protein expression of Myo Va and BMP-2 in melanocytes. Knockdown of BMP-2 by siRNA also affected Myo Va mRNA and protein expression, confirming that MNQO regulates Myo Va through BMP-2. Furthermore, phosphorylation of Smad1/5/8 by BMP2 treatment confirmed that the BMP-2/Smad signaling pathway regulates Myo Va expression in Melan-a melanocytes. Smad-binding elements were found in the Myo Va promoter and phosphorylated Smad1/5/8 bind directly to the Myo Va promoter to activate Myo Va transcription and BMP-2 enhances this binding. These findings provide insight into a new role for BMP-2 in Melan-a melanocytes and a mechanism of regulation of Myo Va expression that may be beneficial in the treatment of albinism or hyperpigmentation disorders., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cellular vesicle long-distance transport along the cytoplasmic actin network has recently been uncovered in several cell systems. In metaphase mouse oocytes, the motor protein myosin-5b (Myo5b) and the actin nucleation factor Spire are recruited to the Rab11a-positive vesicle membrane, forming a ternary complex of Myo5b/Spire/Rab11a that drives the vesicle long-distance transport to the oocyte cortex. However, the mechanism underlying the intermolecular regulation of the Myo5b/Spire/Rab11a complex remains unknown. In this study, we expressed and purified Myo5b, Spire2, and Rab11a proteins, and performed ATPase activity measurements, pulldown and single-molecule motility assays. Our results demonstrate that both Spire2 and Rab11a are required to activate Myo5b motor activity under physiological ionic conditions. The GTBM fragment of Spire2 stimulates the ATPase activity of Myo5b, while Rab11a enhances this activation. This activation occurs by disrupting the head-tail interaction of Myo5b. Furthermore, at the single-molecule level, we observed that the GTBM fragment of Spire2 and Rab11a coordinate to stimulate the Myo5b motility activity. Based on our results, we propose that upon association with the vesicle membrane, Myo5b, Spire2 and Rab11a form a ternary complex, and the inhibited Myo5b is synergistically activated by Spire2 and Rab11a, thereby triggering the long-distance transport of vesicles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Robust linkage between adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The Drosophila multidomain protein Canoe and its mammalian homolog afadin are crucial for this, as in their absence many events of morphogenesis fail. To define the mechanism of action for Canoe, we are taking it apart. Canoe has five folded protein domains and a long intrinsically disordered region. The largest is the Dilute domain, which is shared by Canoe and myosin V. To define the roles of this domain in Canoe, we combined biochemical, genetic and cell biological assays. AlphaFold was used to predict its structure, providing similarities and contrasts with Myosin V. Biochemical data suggested one potential shared function - the ability to dimerize. We generated Canoe mutants with the Dilute domain deleted (CnoΔDIL). Surprisingly, they were viable and fertile. CnoΔDIL localized to adherens junctions and was enriched at junctions under tension. However, when its dose was reduced, CnoΔDIL did not provide fully wild-type function. Furthermore, canoeΔDIL mutants had defects in the orchestrated cell rearrangements of eye development. This reveals the robustness of junction-cytoskeletal connections during morphogenesis and highlights the power of natural selection to maintain protein structure., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Depending on the chemical energy from ATP hydrolysis, myosin V can drive the multistep and continuous coupled cycling process to transport cellular cargo to targeted regions. However, it is still obscure how the molecular memory induced by the multistep coupled transported process could regulate the dynamic behavior of the motor state of myosin V. Here, we propose a novel non-Markovian polymorphic mechanochemical model to investigate the effect of the molecular memory on the mechanic of noise attenuation of myosin V system. We first define an effective transition rate for a multistep coupled reaction process which is the function of memory and system states to transform equivalently the non-Markovian process into the classical Markov process. By noise decomposition technology, it is observed that both the intrinsic and extrinsic noises of the ADP-myosin V bound state (AM ⋅ ADP) exhibit a monotonically decreasing trend with lengthening the molecular memory. Molecular memory as a regulation factor can amplify the contribution of intrinsic noise to the overall noise while reducing the influence of extrinsic noise on the AM ⋅ ADP. Moreover, the modulation of molecular memory could induce stochastic focusing. These results indicate that the role of molecular memory in the myosin V state transition can not only offer a handle to maintain the robustness of the motion system but also serve as a paradigm for studying more complex molecular motors., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microvillus inclusion disease (MVID) is a rare condition that is present from birth and affects the digestive system. People with MVID experience severe diarrhea that is difficult to control, cannot absorb dietary nutrients, and struggle to grow and thrive. In addition, diverse clinical manifestations, some of which are life-threatening, have been reported in cases of MVID. MVID can be caused by variants in the MYO5B, STX3, STXBP2, or UNC45A gene. These genes produce proteins that have been functionally linked to each other in intestinal epithelial cells. MVID associated with STXBP2 variants presents in a subset of patients diagnosed with familial hemophagocytic lymphohistiocytosis type 5. MVID associated with UNC45A variants presents in most patients diagnosed with osteo-oto-hepato-enteric syndrome. Furthermore, variants in MYO5B or STX3 can also cause other diseases that are characterized by phenotypes that can co-occur in subsets of patients diagnosed with MVID. Recent studies involving clinical data and experiments with cells and animals revealed connections between specific phenotypes occurring outside of the digestive system and the type of gene variants that cause MVID. Here, we have reviewed these patterns and correlations, which are expected to be valuable for healthcare professionals in managing the disease and providing personalized care for patients and their families., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of ∼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.

Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Secretory vesicles are often delivered to very specific targets, like pre-synaptic terminals or cell tips, to focus exocytosis. New work suggests that a biomolecular condensate focuses actin filaments that deliver incoming vesicles through the condensate to the plasma membrane., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intestinal enterocytes have an elaborate apical membrane of actin-rich protrusions known as microvilli. The organization of microvilli is orchestrated by the intermicrovillar adhesion complex (IMAC), which connects the distal tips of adjacent microvilli. The IMAC is composed of CDHR2 and CDHR5 as well as the scaffolding proteins USH1C, ANKS4B, and Myosin 7b (MYO7B). To create an IMAC, cells must transport the proteins to the apical membrane. Myosin 5b (MYO5B) is a molecular motor that traffics ion transporters to the apical membrane of enterocytes, and we hypothesized that MYO5B may also be responsible for the localization of IMAC proteins. To address this question, we used two different mouse models: 1 ) neonatal germline MYO5B knockout (MYO5B KO) mice and 2 ) adult intestinal-specific tamoxifen-inducible VillinCre ERT2 ;MYO5B flox/flox mice. In control mice, immunostaining revealed that CDHR2, CDHR5, USH1C, and MYO7B were highly enriched at the tips of the microvilli. In contrast, neonatal germline and adult MYO5B-deficient mice showed loss of apical CDHR2, CDHR5, and MYO7B in the brush border and accumulation in a subapical compartment. Colocalization analysis revealed decreased Mander's coefficients in adult inducible MYO5B-deficient mice compared with control mice for CDHR2, CDHR5, USH1C, and MYO7B. Scanning electron microscopy images further demonstrated aberrant microvilli packing in adult inducible MYO5B-deficient mouse small intestine. These data indicate that MYO5B is responsible for the delivery of IMAC components to the apical membrane. NEW & NOTEWORTHY The intestinal epithelium absorbs nutrients and water through an elaborate apical membrane of highly organized microvilli. Microvilli organization is regulated by the intermicrovillar adhesion complexes, which create links between neighboring microvilli and control microvilli packing and density. In this study, we report a new trafficking partner of the IMAC, Myosin 5b. Loss of Myosin 5b results in a disorganized brush border and failure of IMAC proteins to reach the distal tips of microvilli.

Infantile cholestasis is a common clinical problem in early infancy characterised by impairment in bile formation and/or flow. It requires prompt evaluation for underlying aetiology to initiate appropriate management. Although biliary atresia remains the most important aetiology, metabolic and monogenic disorders are increasingly identified with advances in diagnostic genetic testing. Progressive familial intrahepatic cholestasis disorders characterised by defects in biliary canalicular transport are among the most common monogenic disorders of cholestasis. Homozygous or compound heterozygous mutation in the Myosin 5B gene leading to a progressive familial intrahepatic cholestasis-like phenotype with or without intestinal features of microvillus inclusion disease is a relatively recently identified disorder. The incidence of these newer variants of progressive familial intrahepatic cholestasis is not yet known due to the paucity of studies. We report an uncommon cause of refractory cholestasis reported in a girl who presented with severe pruritus as the primary manifestation.

Myo2, a yeast class V myosin, transports a broad range of organelles and plays important roles in various cellular processes, including cell division in budding yeast. Despite the fact that several structures of Myo2/cargo adaptor complexes have been determined, the understanding of the versatile cargo-binding modes of Myo2 is still very limited, given the large number of cargo adaptors identified for Myo2. Here, we used ColabFold, an AlphaFold2-powered and easy-to-use tool, to predict the complex structures of Myo2-GTD and its several cargo adaptors. After benchmarking the prediction strategy with three Myo2/cargo adaptor complexes that have been determined previously, we successfully predicted the atomic structures of Myo2-GTD in complex with another three cargo adaptors, Vac17, Kar9 and Pea2, which were confirmed by our biochemical characterizations. By systematically comparing the interaction details of the six complexes of Myo2 and its cargo adaptors, we summarized the cargo-binding modes on the three conserved sites of Myo2-GTD, providing an overall picture of the versatile cargo-recognition mechanisms of Myo2. In addition, our study demonstrates an efficient and effective solution to study protein-protein interactions in the future via the AlphaFold2-powered prediction.

E-cadherin has a fundamental role in epithelial tissues by providing cell-cell adhesion. Polarised E-cadherin exocytosis to the lateral plasma membrane is central for cell polarity and epithelial homeostasis. Loss of E-cadherin secretion compromises tissue integrity and is a prerequisite for metastasis. Despite this pivotal role of E-cadherin secretion, the transport mechanism is still unknown. Here we identify Myosin V as the motor for E-cadherin secretion. Our data reveal that Myosin V and F-actin are required for the formation of a continuous apicolateral E-cadherin belt, the zonula adherens. We show by live imaging how Myosin V transports E-cadherin vesicles to the plasma membrane, and distinguish two distinct transport tracks: an apical actin network leading to the zonula adherens and parallel actin bundles leading to the basal-most region of the lateral membrane. E-cadherin secretion starts in endosomes, where Rab11 and Sec15 recruit Myosin V for transport to the zonula adherens. We also shed light on the endosomal sorting of E-cadherin by showing how Rab7 and Snx16 cooperate in moving E-cadherin into the Rab11 compartment. Thus, our data help to understand how polarised E-cadherin secretion maintains epithelial architecture and prevents metastasis., (© 2022 The Authors. Traffic published by John Wiley & Sons Ltd.)

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a rare autosomal recessive condition characterized by a lack of microvilli on the surface of enterocytes, resulting in severe, life-threatening diarrhea that could lead to mortality within the first year of life. We identify two unrelated families, each with one child presenting with severe MVID from birth. Using trio whole-exome sequencing, we observed that the two families share a novel nonsense variant (Glu1589*) in the MYO5B gene, a type Vb myosin motor protein in which rare damaging mutations were previously described to cause MVID. This founder mutation was very rare in public databases and is likely specific to patients of Syrian ancestry. We present a detailed account of both patients' clinical histories to fully characterize the effect of this variant and expand the genotype-phenotype databases for MVID patients from the Middle East., (© 2022 Hassan et al.; Published by Cold Spring Harbor Laboratory Press.)

The maintenance of epithelial architecture necessitates tight regulation of cell size and shape. However, mechanisms underlying epithelial cell size regulation remain poorly understood. We show that the interaction of Myosin Vb with Rab11 prevents the accumulation of apically derived endosomes to maintain cell-size, whereas that with Rab10 regulates vesicular transport from the trans-Golgi. These interactions are required for the fine-tuning of the epithelial cell morphology during zebrafish development. Furthermore, the compensatory cell growth upon cell-proliferation inhibition involves a preferential expansion of the apical domain, leading to flatter epithelial cells, an efficient strategy to cover the surface with fewer cells. This apical domain growth requires post-trans-Golgi transport mediated by the Rab10-interacting Myosin Vb isoform, downstream of the mTOR-Fatty Acid Synthase (FASN) axis. Changes in trans-Golgi morphology indicate that the Golgi synchronizes mTOR-FASN-regulated biosynthetic input and Myosin Vb-Rab10 dependent output. Our study unravels the mechanism of polarized growth in epithelial cells and delineates functions of Myosin Vb isoforms in cell size regulation during development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca 2+ ) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca 2+ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350 nm vs. 1750 nm) transport by myoVa motors. Five thousand simulated liposomes transported within each network adopted one of three states: transport, tug-of-war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug-of-wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, N c . N c is a geometric property of the actin network that depends only on the position and polarity of the actin filaments, transport distance, and the liposome diameter, as evidenced by a fivefold increase in liposome diameter resulting in a fivefold decrease in N c . Thus in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands.

Weibel-Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca 2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation., (© 2022. The Author(s).)

Background & Aims: UNC45A is a myosin (co-)chaperone, and mutations in the UNC45A gene were recently identified in osteo-oto-hepato-enteric (O2HE) syndrome patients presenting with congenital diarrhea and intrahepatic cholestasis. Congenital diarrhea and intrahepatic cholestasis are also the prime symptoms in patients with microvillus inclusion disease (MVID) and mutations in MYO5B, encoding the recycling endosome-associated myosin Vb. The aim of this study was to determine whether UNC45A and myosin Vb are functionally linked., Methods: CRISPR-Cas9 gene editing and site-directed mutagenesis were performed with intestinal epithelial and hepatocellular cell lines, followed by Western blotting, quantitative polymerase chain reaction, and scanning electron and/or confocal fluorescence microscopy to determine the relationship between (mutants of) UNC45A and myosin Vb., Results: UNC45A depletion in intestinal and hepatic cells reduced myosin Vb protein expression, and in intestinal epithelial cells, it affected 2 myosin Vb-dependent processes that underlie MVID pathogenesis: rat sarcoma-associated binding protein (RAB)11A-positve recycling endosome positioning and microvilli development. Reintroduction of UNC45A in UNC45A-depleted cells restored myosin Vb expression, and reintroduction of UNC45A or myosin Vb, but not the O2HE patient UNC45A-c.1268T>A variant, restored recycling endosome positioning and microvilli development. The O2HE patient-associated p.V423D substitution, encoded by the UNC45A-c.1268T>A variant, impaired UNC45A protein stability but as such not the ability of UNC45A to promote myosin Vb expression and microvilli development., Conclusions: A functional relationship exists between UNC45A and myosin Vb, thereby connecting 2 rare congenital diseases with overlapping enteropathy at the molecular level. Protein instability rather than functional impairment underlies the pathogenicity of the O2HE syndrome-associated UNC45A-p.V423D mutation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Microvillus inclusion disease (MVID) is a congenital diarrheal disorder resulting in life-threatening secretory diarrhea in newborns. Inactivating and nonsense mutations in myosin Vb (MYO5B) have been identified in MVID patients. Work using patient tissues, cell lines, mice, and pigs has led to critical insights into the pathology of MVID and a better understanding of both apical trafficking in intestinal enterocytes and intestinal stem cell differentiation. These studies have demonstrated that loss of MYO5B or inactivating mutations lead to loss of apical sodium and water transporters, without loss of apical CFTR, accounting for the major pathology of the disease. In addition, loss of MYO5B expression induces the formation of microvillus inclusions through apical bulk endocytosis that utilizes dynamin and PACSIN2 and recruits tight junction proteins to the sites of bulk endosome formation. Importantly, formation of microvillus inclusions is not required for the induction of diarrhea. Recent investigations have demonstrated that administration of lysophosphatidic acid (LPA) can partially reestablish apical ion transporters in enterocytes of MYO5B KO mice. In addition, further studies have shown that MYO5B loss induces an imbalance in Wnt/Notch signaling pathways that can lead to alterations in enterocyte maturation and tuft cell lineage differentiation. Inhibition of Notch signaling leads to improvements in those cell differentiation deficits. These studies demonstrate that directed strategies through LPA receptor activation and Notch inhibition can bypass the inhibitory effects of MYO5B loss. Thus, effective strategies may be successful in MVID patients and other congenital diarrhea syndromes to reestablish proper apical membrane absorption of sodium and water in enterocytes and ameliorate life-threatening congenital diarrhea., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A major question in cell biology is, how are organelles and macromolecular machines moved within a cell? The delivery of cargoes to the right place at the right time within a cell is critical to cellular health. Failure to do so is often catastrophic for animal physiology and results in diseases of the gut, brain, and skin. In budding yeast, a myosin V motor, Myo2, moves cellular materials from the mother cell into the growing daughter bud. Myo2-based transport ensures that cellular contents are shared during cell division. During transport, Myo2 is often linked to its cargo via cargo-specific adaptor proteins. This simple organism thus serves as a powerful tool to study how myosin V moves cargo, such as organelles. Some critical questions include how myosin V moves along the actin cytoskeleton, or how myosin V attaches to cargo in the mother. Other critical questions include how the cargo is released from myosin V when it reaches its final destination in the bud. Here, we review the mechanisms that regulate the vacuole-specific adaptor protein, Vac17, to ensure that Myo2 delivers the vacuole to the bud and releases it at the right place and the right time. Recent studies have revealed that Vac17 is regulated by ubiquitylation and phosphorylation events that coordinate its degradation and the detachment of the vacuole from Myo2. Thus, multiple post-translational modifications tightly coordinate cargo delivery with cellular events. It is tempting to speculate that similar mechanisms regulate other cargoes and molecular motors., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The directional movement and positioning of organelles and macromolecules is essential for regulating and maintaining cellular functions in eukaryotic cells. In plants, these processes are actin-based and driven by class XI myosins, which transport various cargos in a directed manner. As the analysis of myosin function is challenging due to high levels of redundancy, dominant negative acting truncated myosins have frequently been used to study intracellular transport processes. A comparison of the dominant negative effect of the coiled-coil domains and the GTD domains revealed a much stronger inhibition of P-body movement by the GTD domains. In addition, we show that the GTD domain does not inhibit P-body movement when driven by a hybrid myosin in which the GTD domain was replaced by DCP2. These data suggest that the dominant negative effect of myosin tails involves a competition of the GTD domains for cargo binding sites., Competing Interests: The authors have declared that no competing interests exist.

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape., (© 2021 Johnsson et al.)

Retrieval of cargo proteins from the endosome towards the trans-Golgi network (TGN) is a crucial intracellular process for cellular homeostasis. Its dysfunction is associated with pathogenesis of Alzheimer and Parkinson's diseases. Myosin family proteins are cellular motors walking along actin filaments by utilizing the chemical energy from ATP hydrolysis, known to involve in pleiotropic cellular trafficking pathways. However, the question of whether myosins play a role in the trafficking of Snc1 and Vps10 has not been addressed yet. The present study assesses the potential roles of all five yeast myosins in the recycling of two membrane cargo, Snc1 and Vps10. It appears that all myosins except Myo2 are not required for the Snc1 traffic, while it was found that Myo1 and 2 play important roles for Vps10 retrieval from the endosome and the vacuole. Multiple myo2 mutants harboring a point mutation in the actin binding or the cargo binding tail domain were characterized to demonstrate abnormal Vps10-GFP and GFP-Snc1 distribution phenotypes, suggesting a severe defect in their sorting and trafficking at the endosome. Furthermore, Vps10-GFP patches in all tested myo2 mutants were found to be near stationary with quantitative live cell imaging. Finally, we found that actin cables in the myo2 mutant cells were considerably disrupted, which may aggravate the trafficking of Vps10 from the endosome. Together, our results provide novel insights into the function of Myo-family proteins in the recycling traffic of Vps10 and Snc1 destined for the TGN., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Activating mutations in LRRK2 kinase causes Parkinson's disease. Pathogenic LRRK2 phosphorylates a subset of Rab GTPases and blocks ciliogenesis. Thus, defining novel phospho-Rab interacting partners is critical to our understanding of the molecular basis of LRRK2 pathogenesis. RILPL2 binds with strong preference to LRRK2-phosphorylated Rab8A and Rab10. RILPL2 is a binding partner of the motor protein and Rab effector, Myosin Va. We show here that the globular tail domain of Myosin Va also contains a high affinity binding site for LRRK2-phosphorylated Rab10. In the presence of pathogenic LRRK2, RILPL2 and MyoVa relocalize to the peri-centriolar region in a phosphoRab10-dependent manner. PhosphoRab10 retains Myosin Va over pericentriolar membranes as determined by fluorescence loss in photobleaching microscopy. Without pathogenic LRRK2, RILPL2 is not essential for ciliogenesis but RILPL2 over-expression blocks ciliogenesis in RPE cells independent of tau tubulin kinase recruitment to the mother centriole. These experiments show that LRRK2 generated-phosphoRab10 dramatically redistributes a significant fraction of Myosin Va and RILPL2 to the mother centriole in a manner that likely interferes with Myosin Va's role in ciliogenesis., (© 2021 Dhekne et al.)

A major question in cell biology is, how are organelles and large macromolecular complexes transported within a cell? Myosin V molecular motors play critical roles in the distribution of organelles, vesicles, and mRNA. Mis-localization of organelles that depend on myosin V motors underlie diseases in the skin, gut, and brain. Thus, the delivery of organelles to their proper destination is important for animal physiology and cellular function. Cargoes attach to myosin V motors via cargo specific adaptor proteins, which transiently bridge motors to their cargoes. Regulation of these adaptor proteins play key roles in the regulation of cargo transport. Emerging studies reveal that cargo adaptors play additional essential roles in the activation of myosin V, and the regulation of actin filaments. Here, we review how motor-adaptor interactions are controlled to regulate the proper loading and unloading of cargoes, as well as roles of adaptor proteins in the regulation of myosin V activity and the dynamics of actin filaments., (Copyright © 2021. Published by Elsevier Ltd.)

Background & Aims: The molecular motor, Myosin Vb (MYO5B), is well documented for its role in trafficking cargo to the apical membrane of epithelial cells. Despite its involvement in regulating apical proteins, the role of MYO5B in cell polarity is less clear. Inactivating mutations in MYO5B result in microvillus inclusion disease (MVID), a disorder characterized by loss of key apical transporters and the presence of intracellular inclusions in enterocytes. We previously identified that inclusions in Myo5b knockout (KO) mice form from invagination of the apical brush border via apical bulk endocytosis. Herein, we sought to elucidate the role of polarity complexes and tight junction proteins during the formation of inclusions., Methods: Intestinal tissue from neonatal control and Myo5b KO littermates was analyzed by immunofluorescence to determine the localization of polarity complexes and tight junction proteins., Results: Proteins that make up the apical polarity complexes-Crumbs3 and Pars complexes-were associated with inclusions in Myo5b KO mice. In addition, tight junction proteins were observed to be concentrated over inclusions that were present at the apical membrane of Myo5b-deficient enterocytes in vivo and in vitro. Our mouse findings are complemented by immunostaining in a large animal swine model of MVID genetically engineered to express a human MVID-associated mutation that shows an accumulation of Claudin-2 over forming inclusions. The findings from our swine model of MVID suggest that a similar mechanism of tight junction accumulation occurs in patients with MVID., Conclusions: These data show that apical bulk endocytosis involves the altered localization of apical polarity proteins and tight junction proteins after loss of Myo5b., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport., (Copyright © 2020 © 2020 Gunther et al. Published by Elsevier Inc. All rights reserved.)

Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β-cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric guanosine-5'-triphosphatase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Melanophilin-mutated leaden mouse and melanophilin-downregulated human pancreatic β-cells both exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca 2+ ] i rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane., (© 2020 by the American Diabetes Association.)

Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Overexpression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR (Fes/CIP4 homology-Bin-Amphiphysin-Rvs protein)-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 overexpression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP 2 -enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho state, and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.

In hippocampal pyramidal cells, a small subset of dendritic spines contain endoplasmic reticulum (ER). In large spines, ER frequently forms a spine apparatus, while smaller spines contain just a single tubule of smooth ER. Here we show that the ER visits dendritic spines in a non-random manner, targeting spines during periods of high synaptic activity. When we blocked ER motility using a dominant negative approach against myosin V, spine synapses became stronger compared to controls. We were not able to further potentiate these maxed-out synapses, but long-term depression (LTD) was readily induced by low-frequency stimulation. We conclude that the brief ER visits to active spines have the important function of preventing runaway potentiation of individual spine synapses, keeping most of them at an intermediate strength level from which both long-term potentiation (LTP) and LTD are possible.

Cilia and flagella are microtubule-based cellular projections with important sensory and motility functions. Their absence or malfunction is associated with a growing number of human diseases collectively referred to as ciliopathies. However, the fundamental mechanisms underpinning cilia biogenesis and functions remain only partly understood. Here, we show that depleting LUZP1 or its interacting protein, EPLIN, increases the levels of MyosinVa at the centrosome and primary cilia formation. We further show that LUZP1 localizes to both actin filaments and the centrosome/basal body. Like EPLIN, LUZP1 is an actin-stabilizing protein that regulates actin dynamics, at least in part, by mobilizing ARP2 to the centrosomes. Both LUZP1 and EPLIN interact with known ciliogenesis and cilia-length regulators and as such represent novel players in actin-dependent centrosome to basal body conversion. Ciliogenesis deregulation caused by LUZP1 or EPLIN loss may thus contribute to the pathology of their associated disease states., (© 2020 Gonçalves et al.)

The human fungal pathogen Aspergillus fumigatus causes life-threatening invasive aspergillosis in immunocompromised individuals. Adaptation to the host environment is integral to survival of A. fumigatus and requires the coordination of short- and long-distance vesicular transport to move essential components throughout the fungus. We previously reported the importance of MyoE, the only class V myosin, for hyphal growth and virulence of A. fumigatus. Class V myosins are actin-based, cargo-carrying motor proteins that contain unique binding sites for specific cargo. Specific cargo carried by myosin V has not been identified in any fungus, and previous studies have only identified single components that interact with class V myosins. Here we utilized a mass spectrometry-based whole proteomic approach to identify MyoE interacting proteins in A. fumigatus for the first time. Several proteins previously shown to interact with myosin V through physical and genetic approaches were confirmed, validating our proteomic analysis. Importantly, we identified novel MyoE-interacting proteins, including members of the cytoskeleton network, cell wall synthesis, calcium signaling and a group of coat protein complex II (COPII) proteins involved in the endoplasmic reticulum (ER) to Golgi transport. Furthermore, we analyzed the localization patterns of the COPII proteins, UsoA (Uso1), SrgE (Sec31), and SrgF (Sec23), which suggested a potential role for MyoE in ER to Golgi trafficking., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Spitzenkörper (SPK) constitutes a collection of secretory vesicles and polarity-related proteins intimately associated with polarized growth of fungal hyphae. Many SPK-localized proteins are known, but their assembly and dynamics remain poorly understood. Here, we identify protein-protein interaction cascades leading to assembly of two SPK scaffolds and recruitment of diverse effectors in Neurospora crassa. Both scaffolds are transported to the SPK by the myosin V motor (MYO-5), with the coiled-coil protein SPZ-1 acting as cargo adaptor. Neither scaffold appears to be required for accumulation of SPK secretory vesicles. One scaffold consists of Leashin-2 (LAH-2), which is required for SPK localization of the signalling kinase COT-1 and the glycolysis enzyme GPI-1. The other scaffold comprises a complex of Janus-1 (JNS-1) and the polarisome protein SPA-2. Via its Spa homology domain (SHD), SPA-2 recruits a calponin domain-containing F-actin effector (CCP-1). The SHD NMR structure reveals a conserved surface groove required for effector binding. Similarities between SPA-2/JNS-1 and the metazoan GIT/PIX complex identify foundational features of the cell polarity apparatus that predate the fungal-metazoan divergence.

Telomere-led rapid chromosome movements or rapid prophase movements direct fundamental meiotic processes required for successful haploidization of the genome. Critical components of the machinery that generates rapid prophase movements are unknown, and the mechanism underlying rapid prophase movements remains poorly understood. We identified S. cerevisiae Mps2 as the outer nuclear membrane protein that connects the LINC complex with the cytoskeleton. We also demonstrate that the motor Myo2 works together with Mps2 to couple the telomeres to the actin cytoskeleton. Further, we show that Csm4 interacts with Mps2 and is required for perinuclear localization of Myo2, implicating Csm4 as a regulator of the Mps2-Myo2 interaction. We propose a model in which the newly identified functions of Mps2 and Myo2 cooperate with Csm4 to drive chromosome movements in meiotic prophase by coupling telomeres to the actin cytoskeleton., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. We use the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins - Merlin, Moesin and Expanded - whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Local accumulation of oskar (osk ) mRNA in the Drosophila oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for osk mRNA posterior localization. In this study, we use Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for osk mRNA. We demonstrate that posterior localization of osk /Staufen is determined by competition between kinesin-1 and myosin-V. While kinesin-1 removes osk /Staufen from the cortex along microtubules, myosin-V anchors osk /Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of Drosophila oocytes is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density., Competing Interests: WL, ML, RL, NB, AR, MG, JS, VG No competing interests declared

Vertebrates usually have three class V myosin paralogues (MyoV) to control membrane trafficking in the actin-rich cell cortex, but their functional overlapping or differentiation through cargoes selectivity is yet only partially understood. In this work, we reveal that the globular tail domain of MyoVc binds to the active form of small GTPase Rab3A with nanomolar affinity, a feature shared with MyoVa but not with MyoVb. Using molecular docking analyses guided by chemical cross-linking restraints, we propose a model to explain how Rab3A selectively recognizes MyoVa and MyoVc via a distinct binding site from that used by Rab11A. The MyoVa/c binding interface involves multiple residues from both lobules (I and II) and the short helix at the α2-α3 link region, which is conserved between MyoVa and MyoVc, but not in MyoVb. This motif is also responsible for the selective binding of RILPL2 by MyoVa and potentially MyoVc. Together, these findings support the selective recruitment of MyoVa and MyoVc to exocytic pathways via Rab3A and expand our knowledge about the functional evolution of class V myosins. SIGNIFICANCE: Hormone secretion, neurotransmitter release, and cytoplasm membrane recycling are examples of processes that rely on the interaction of molecular motors and Rab GTPases to regulate the intracellular trafficking and tethering of vesicles. Defects in these proteins may cause neurological impairment, immunodeficiency, and other severe disorders, being fatal in some cases. Despite their crucial roles, little is known about how these molecular motors are selectively recruited by specific members of the large family of Rab GTPases. In this study, we unveil the interaction between the actin-based molecular motor Myosin Vc and the small GTPase Rab3A, a key coordinator of vesicle trafficking and exocytosis in mammalian cells. Moreover, we propose a model for their recognition and demonstrate that Rab3A specifically binds to the globular tail of Myosins Va and Vc, but not of Myosin Vb, advancing our knowledge about the molecular basis for the selective recruitment of class V myosins by Rab GTPases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The molecular motor myosin V transports cargo by stepping on actin filaments, executing a random diffusive search for actin binding sites at each step. A recent experiment suggests that the joint between the myosin lever arms may not rotate freely, as assumed in earlier studies, but instead has a preferred angle giving rise to structurally constrained diffusion. We address this controversy through comprehensive analytical and numerical modeling of myosin V diffusion and stepping. When the joint is constrained, our model reproduces the experimentally observed diffusion, allowing us to estimate bounds on the constraint energy. We also test the consistency between the constrained diffusion model and previous measurements of step size distributions and the load dependence of various observable quantities. The theory lets us address the biological significance of the constrained joint and provides testable predictions of new myosin behaviors, including the stomp distribution and the run length under off-axis force., Competing Interests: DH, RT, MH, DT No competing interests declared, (© 2020, Hathcock et al.)

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting β Heavy -Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P 2 and formation of a novel, Moesin- and PI(4,5)P 2 -enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland., Competing Interests: JL, WL, MB, DF No competing interests declared, EK Reviewing editor, eLife, (© 2019, Lattner et al.)