Microtubules are part of the dynamic cytoskeleton network and composed of tubulin dimers. They are the main tracks used in cells to organize organelle positioning and trafficking of cargos. In this ...Review, we compile recent findings on the involvement of microtubules in anterograde protein transport. First, we highlight the importance of microtubules in organelle positioning. Second, we discuss the involvement of microtubules within different trafficking steps, in particular between the endoplasmic reticulum and the Golgi complex, traffic through the Golgi complex itself and in post-Golgi processes. A large number of studies have assessed the involvement of microtubules in transport of cargo from the Golgi complex to the cell surface. We focus here on the role of kinesin motor proteins and protein interactions in post-Golgi transport, as well as the impact of tubulin post-translational modifications. Last, in light of recent findings, we highlight the role microtubules have in exocytosis, the final step of secretory protein transport, occurring close to focal adhesions.
Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma ...membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.
Abstract
Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what ...extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.
Distinct PSD-95 clusters are primary landmarks of postsynaptic densities (PSDs), which are specialized membrane regions for synapses. However, the mechanism that defines the locations of PSD-95 ...clusters and whether or how they are reorganized inside individual dendritic spines remains controversial. Because palmitoylation regulates PSD-95 membrane targeting, we combined a conformation-specific recombinant antibody against palmitoylated PSD-95 with live-cell super-resolution imaging and discovered subsynaptic nanodomains composed of palmitoylated PSD-95 that serve as elementary units of the PSD. PSD-95 in nanodomains underwent continuous de/repalmitoylation cycles driven by local palmitoylating activity, ensuring the maintenance of compartmentalized PSD-95 clusters within individual spines. Plasma membrane targeting of DHHC2 palmitoyltransferase rapidly recruited PSD-95 to the plasma membrane and proved essential for postsynaptic nanodomain formation. Furthermore, changes in synaptic activity rapidly reorganized PSD-95 nano-architecture through plasma membrane-inserted DHHC2. Thus, the first genetically encoded antibody sensitive to palmitoylation reveals an instructive role of local palmitoylation machinery in creating activity-responsive PSD-95 nanodomains, contributing to the PSD (re)organization.
Ephrins can elicit either contact-mediated cell-cell adhesion or repulsion, depending on the efficiency of the removal of their ligand-receptor complexes from the cell surface, thus controlling ...tissue morphogenesis and oncogenic development. However, the dynamic of the turnover of newly assembled ephrin-Eph complexes during cell-cell interactions remains mostly unexplored. Here, we show that ephrin-A1-EphA2 complexes are locally formed at the tip of the filopodia, at cell-to-cell contacts. Clusters of ephrin-A1 from donor cells surf on filopodia associated to EphA2-bearing subdomains of acceptor cells. Full-length ephrin-A1 is transferred to acceptor cells by trans-endocytosis through a proteolysis-independent mechanism. Trans-endocytosed ephrin-A1 bound to its receptor enables signaling to be emitted from endo-lysosomes of acceptor cells. Localized trans-endocytosis of ephrin-A1 sustains contact-mediated repulsion on cancer cells. Our results uncover the essential role played by local concentration at the tip of filopodia and the trans-endocytosis of full-length ephrin to maintain long-lasting ephrin signaling.
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•Ephrin-A1 trafficking synchronization allows visualization of its transport to filopodia•Signaling occurs at the tips of filopodia•Ephrin-1-EphA2 interaction induces trans-endocytosis of the complex•Trans-endocytosis enables long-term signaling from lysosomes
Ephrins can elicit either cell-cell adhesion or repulsion. Using a synchronized transport assay, Valenzuela and Perez show that clusters of ephrin-A1 from donor cells surf on filopodia associated to the receptor EphA2 on acceptor cells. Full-length ephrin-A1 is then transferred to acceptor cells by trans-endocytosis through a proteolysis-independent mechanism.
Microtubules are hollow tubes essential for many cellular functions such as cell polarization and migration, intracellular trafficking and cell division. They are polarized polymers composed of α and ...β tubulin that are, in most cells, nucleated at the centrosome at the center of the cell. Microtubule plus-ends are oriented towards the periphery of the cell and explore the cytoplasm in a very dynamic manner. Microtubule alternate between phases of growth and shrinkage in a manner described as dynamic instability. Their dynamics is highly regulated by multiple factors: tubulin post-translational modifications such as detyrosination or acetylation, and microtubule-associated proteins, among them the plus-tip tracking proteins. This regulation is necessary for microtubule functions in the cell. In this review, we will focus on the role of microtubules in intracellular organization. After an overview of the mechanisms responsible for the regulation of microtubule dynamics, the major roles of microtubules dynamics in organelle positioning and organization in interphase cells will be discussed. Conversely, the role of certain organelles, like the nucleus and the Golgi apparatus as microtubule organizing centers will be reviewed. We will then consider the role of microtubules in the establishment and maintenance of cell polarity using few examples of cell polarization: epithelial cells, neurons and migrating cells. In these cells, the microtubule network is reorganized and undergoes specific and local regulation events; microtubules also participate in the intracellular reorganization of different organelles to ensure proper cell differentiation.
Membrane damage is a daily threat to the life of a cell, especially cells from muscles, gut, epidermis and vasculature, tissues that are particularly subjected to mechanical stress. Damages can come ...from different sources and give rise to different holes in terms of size and nature. For example, while some holes are simply scratches in the lipid bilayer, others are delimited by pore forming proteins. It is thus expectable that these wounds will not evolve similarly in a cellular context, and that repair mechanisms will differ to a certain extent. It would therefore be misleading to fully generalize cell membrane damage and repair, and consider it as one universal phenomenon. Indeed, damage has been observed in cells ranging from the rather small mammalian cells (∼30μm) to the very big Urchin egg (∼100μm). Moreover, the wounds observed or artificially induced in eukaryotic cells range from some nanometers to several micrometers, and can be delimited by particular molecules as mentioned before. This chapter aims at reviewing the different physico-chemical and biological parameters that can influence wound evolution in cells and to conciliate the different repair mechanisms that have been described by evaluating them in their cellular and wound type context.
Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 ...binding sites, and selectively accumulates at the tips of axons. However, it remains unknown what cue KIF5 recognizes to result in this selective accumulation. We found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.
To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are ...transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.
In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single ...domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.
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