This work uncovered an unprecedented role of V-ATPase in shaping the macropinocytic cup in the pathogenic amoeba Entamoeba histolytica and highlights the novel role of V-ATPase in directly driving actin polymerization, in conjunction with phosphatidic acid, to shape the macropinocytic cup.
Key mechanistic steps coordinating ATP-driven MreB polymerization and turnover are established and provide a basis for a complete MreB assembly–disassembly cycle and for further elucidating how MreB dynamics contribute to cell-wall organization.
Alba de San Eustaquio-Campillo, C. Cornilleau, Sana Afensiss et al.· bioRxiv· 0 citations
Vacuolar H+ ATPases (v-ATPases) are conserved proton pumps that support diverse biological functions through acidification of cellular organelles. The protozoan parasite Leishmania requires its v-ATPase for survival in the sand fly vector and mammalian host, but genetic mutants remain viable in vitro. To gain further insight into this conditionally lethal phenotype, we first mapped organellar localization of the v-ATPase by co-localisation imaging of fluorescently tagged v-ATPase subunits and organelle markers. The v-ATPase signal was strongest in the flagellar pocket region, consistent with enrichment in the contractile vacuole complex (CVC). To define the conditions that require a functional v-ATPase, deletion mutants were exposed to different stresses (pH, temperature, osmolality, dense culture). All tested deviations from standard culture conditions affected the mutants’ growth rate, viability or both. Despite differences in phenotype severity, all stressors triggered the formation of a large autolysosome, positive for the autophagy marker protein ATG8 and the lysosomal enzyme cysteine peptidase A, indicating an arrest at the final step of autophagy. Measurements with the pH sensor pHLuorin2 showed that the luminal pH of the lysosomes was 5.6 in unperturbed promastigotes and 7.1 in v-ATPase mutants. These data support a canonical function for the Leishmania v-ATPase in lysosome acidification and autophagy, which is essential for parasite differentiation, and identify the poorly characterized Leishmania CVC as another major site of v-ATPase concentration.
Çağla Alagöz, Andreia Albuquerque-Wendt, E. Gluenz· bioRxiv· 0 citations
Ring-like AAA+ (ATPases Associated with diverse cellular Activities) biological machines mediate protein remodeling to assist a broad range of essential cellular functions. The nucleotide-dependent remodeling action involves intra- and inter-ring allosteric communication to generate mechanical force applied onto the substrate by a set of loops that protrude into the central channel. In this study, we probe these allosteric mechanisms through a comparative study of the katanin, a microtubule severing protein including a clade 3 AAA domain, and the double-ring ClpB, a protein disaggregase including both a clade 3 and a clade 5 AAA domain. Our molecular dynamics simulations, combined with machine learning and bioinformatic analysis, reveal both similar mechanisms involving the clade 3 domain and ClpB-specific ones involving communication with the clade 5 domain. We find that both nucleotide and substrate polypeptide binding restrict the conformational landscape sampled by katanin and ClpB, with ligand-specific conformations observed in the latter case. Allosteric contributions of secondary structure elements, ranked by using SHapley Additive exPlanations analysis in machine learning approaches and binary classification of features in ligand states, highlight the important role of regions adjacent to the nucleotide-binding site and the pore loops. Amino acid-level analysis of the allosteric paths reveals that intra-ring cooperativity modulates long-distance communication within the AAA+ protomers.
B-cells extract immobilized antigens via the immune synapse (IS), a specialized, mechanically regulated interface that integrates receptor signaling with dynamic cytoskeletal remodeling. At this interface, actin polymerization, cortical tension, and membrane trafficking cooperate to generate the forces required for antigen extraction. Although actin dynamics are known to be essential for these processes, the molecularmechanisms that organize the cytoskeletal architecture underlying B-cell mechanosensing remain incompletely understood.Here, we identify SWAP70 as a central regulator of cytoskeletal organization at the IS. We show that SWAP70 is rapidly recruited to regions of intense actin remodeling at the synaptic interface, where it forms a stable network with slower turnover than filamentous actin. Importantly, this stability depends on formin activity, indicating that SWAP70 cooperates with the linear actin filament to help stabilize the cortical cytoskeleton.Functional analyses demonstrate that SWAP70 is not required for global actin turnover but instead regulates higher-order cytoskeletal organization. Loss of SWAP70 leads to pronounced alterations in synapse architecture, including increased cell spreading, reduced actin retrograde flow, and enhanced formation of actin foci. Despite these structural changes, actin turnover, as measured by FRAP, remains largely unchanged, supporting the idea that SWAP70 primarily controls the spatial organization and mechanical integration of actin networks rather than their polymerization kinetics. These cytoskeletal defects are accompanied by major changes in IS organization. In the absence of SWAP70, B cells display increased numbers of B cell receptor (BCR) clusters and enhanced recruitment of lysosomes to the IS. Additionally, these cells upregulate the amount of clathrin-coated pits and thereby exhibit increased antigen extraction.Our findings identify SWAP70 as a key organizer of cytoskeletal architecture that constrains the mechanical and trafficking responses of B-cells during IS formation. Given that SWAP70 deficiency has been associated with autoimmune phenotypes, we propose that SWAP70-dependent regulation of cytoskeletal architecture at the IS serves as a critical checkpoint that prevents aberrant B-cell activationand helps maintain immune tolerance.
The intracellular cilia assembly pathway is a complex, multistep process that requires the continuous and coordinated incorporation of membrane material. However, how membrane remodeling occurs during early ciliogenesis is not yet understood. Moreover, the identity of the organelle(s) that supply membrane material for the nascent cilium has yet to be determined. Here, we extend the current model of primary cilia formation by showing that randomly attached distal appendage vesicles and tubules fuse laterally to generate a doughnut-shaped membrane structure. Centripetal fusion events follow to close the central hole. Our data demonstrate that both the Golgi apparatus and endocytotic pathways independently contribute to ciliogenesis. We identify the endocytotic protein GRAF1 as being essential during the early stages of ciliogenesis and for the delivery of plasma membrane-derived material to the developing ciliary membrane. Our three-dimensional ultrastructural analysis uncovers previously unrecognized intermediate stages in the intracellular cilia assembly pathway with GRAF1 as a regulator of ciliogenesis. A three-dimensional ultrastructural analysis reveals previously unrecognized intermediate stages in the formation of primary cilia. It is demonstrated that both the endocytotic protein GRAF1 and the Golgi apparatus contribute to the intracellular pathway of ciliogenesis.
Kerstin N. Schmidt, Korbinian Buerger, Olga Maier et al.· Nature Communications· 0 citations
Directional transport of massive secretory vesicles to the germination site is a key process for pollen germination, but the molecular mechanism remains unclear. Here, we find that Myo11C proteins, Myo11C1 and Myo11C2, act redundantly. This functional redundancy is evidenced by impaired vesicle tethering efficiency and aberrant F-actin organization in myo11c1 myo11c2 double mutant. Myo11C1 unexpectedly colocalized with AtFH5-located secretory vesicles (AtFH5-SVs), rather than with actin filaments. Simulated modeling and truncation mutation analyses show that Myo11C binds actin filaments nucleated from AtFH5-SVs and exhibits plus-end–directed motility, leading to its polarized localization at F-actin plus-ends. Upon dissociating from F-actin, Myo11C directly interacts with AtFH5-SVs to promote their aggregation. E447 is identified as a critical residue for Myo11C1 binding to F-actin, while R1377 and R1452 are conserved residues essential for Myo11C1’s interaction with secretory vesicle–located SEC5B. Together, our work uncovers the molecular basis by which formin-actin-myosin working system generates force driving massive secretory vesicles for directional transport during pollen germination.