Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 towards the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4, but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis. [Media: see text] [Media: see text]

A novel RAB11-containing adaptor complex anchoring myosin-5 to secretory vesicles

Hyphal fungi grow rapidly by apical extension, providing a notorious example of polarized growth. The continuous supply of secretory vesicles necessary to meet the demands of the extending tip and the long intracellular distances existing between the tip and the basal septum, often localized > 100 µm away from the former, impose the need of efficient networks of intracellular traffic involving exquisite cooperation between microtubule- and actin-mediated transport. In Aspergillus nidulans kinesin-1 conveys secretory vesicles to the hyphal tip, where they are transferred to myosin-5, which focuses them at the growing apex, thereby determining cell shape. This relay mechanism and the central role played by myosin-5 in hyphal morphogenesis suggested that the mechanisms anchoring secretory vesicles to this motor should involve specific adaptor(s) ensuring the robustness of actomyosin-dependent transport.Secretory vesicles are charged with RAB11, a regulatory GTPase that determines the Golgi to post-Golgi identity transition. By using a combination of shotgun proteomics, GST-RAB pull-down assays, in vitro reconstitution experiments, targeted reverse genetics and multidimensional fluorescence microscopy with endogenously tagged proteins we show that RAB11, the master regulator of fungal exocytosis, mediates myosin-5 engagement both by contacting the motor and by recruiting UDS1, a homologue of an as yet uncharacterized Schizosaccharomyces protein ‘upregulated during mitosis’, which we demonstrate to be a novel RAB11 effector. Analytical ultracentrifugation determined that UDS1 is an elongated dimer and negative-stain electron microscopy showed that, in agreement, UDS1 is rod-shaped. UDS1 does not contact myosin-5 directly, but rather recruits the coiled-coil HMSV, which bridges RAB11/UDS1 to myosin-5. An HMSV-scaffolded complex containing UDS1 and myosin-5 is present in cells, and a RAB11-UDS1-HMSV complex can be reconstituted in vitro in a RAB nucleotide state-dependent manner. In the absence of UDS1/HMSV the steady state levels of myosin-5 at the apical vesicle supply center diminish markedly, such that microtubule-dependent transport spreading vesicles across the apical dome predominates over apex-focused actin-mediated transport. As a consequence, RAB11 and chitin-synthase B (a cargo of the RAB11 pathway) are not focused at the apex, being distributed instead across the apical dome. Therefore, the RAB11 effector UDS1/HMSV cooperates with the GTPase to adapt secretory vesicles to myosin-5, which is required for the apical targeting of RAB11 cargoes and thus for the normal morphology of the hyphae.

Knockin’ on Egg’s Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype–phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

Orchestrated Restructuring Events During Secretory Granule Maturation Mediate Intragranular Cargo Segregation

Regulated secretion is an essential process where proteins are packaged into membranous secretory vesicles. However, the details of cargo packaging and secretory granule maturation are largely unknown. Here, we demonstrate that multiple distinct proteins undergo orchestrated intragranular restructuring during secretory granule maturation in vivo, to allow spatial segregation of distinct components within the same granule. Furthermore, through a combination of genetics and multimodality imaging, we demonstrate the molecular identity of each distinct intragranular structure. We further identify genes that are essential for the temporally-ordered restructuring events, including those controlling pH (vha16.1), Cl- ions (Clic and ClC-c) and Ca2+ ions (fwe). Finally, we show that altered cargo glycosylation influences dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular, segregation of cargo through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct proteins are efficiently packaged into and secreted from the same secretory granule may provide insight into diseases resulting from defects in secretion.

Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.

Immune Cell Degranulation in Fungal Host Defence

Humans have developed complex immune systems that defend against invading microbes, including fungal pathogens. Many highly specialized cells of the immune system share the ability to store antimicrobial compounds in membrane bound organelles that can be immediately deployed to eradicate or inhibit growth of invading pathogens. These membrane-bound organelles consist of secretory vesicles or granules, which move to the surface of the cell, where they fuse with the plasma membrane to release their contents in the process of degranulation. Lymphocytes, macrophages, neutrophils, mast cells, eosinophils, and basophils all degranulate in fungal host defence. While anti-microbial secretory vesicles are shared among different immune cell types, information about each cell type has emerged independently leading to an uncoordinated and confusing classification of granules and incomplete description of the mechanism by which they are deployed. While there are important differences, there are many similarities in granule morphology, granule content, stimulus for degranulation, granule trafficking, and release of granules against fungal pathogens. In this review, we describe the similarities and differences in an attempt to translate knowledge from one immune cell to another that may facilitate further studies in the context of fungal host defence.

An Examination of the Putative Role of Melatonin in Exosome Biogenesis

During the last two decades, melatonin has been found to have pleiotropic effects via different mechanisms on its target cells. Data are abundant for some aspects of the signaling pathways within cells while other casual mechanisms have not been adequately addressed. From an evolutionary perspective, eukaryotic cells are equipped with a set of interrelated endomembrane systems consisting of intracellular organelles and secretory vesicles. Of these, exosomes are touted as cargo-laden secretory vesicles that originate from the endosomal multivesicular machinery which participate in a mutual cross-talk at different cellular interfaces. It has been documented that cells transfer various biomolecules and genetic elements through exosomes to sites remote from the original cell in a paracrine manner. Findings related to the molecular mechanisms between melatonin and exosomal biogenesis and cargo sorting are the subject of the current review. The clarification of the interplay between melatonin and exosome biogenesis and cargo sorting at the molecular level will help to define a cell’s secretion capacity. This review precisely addresses the role and potential significance of melatonin in determining the efflux capacity of cells via the exosomal pathway. Certain cells, for example, stem cells actively increase exosome efflux in response to melatonin treatment which accelerates tissue regeneration after transplantation into the injured sites.