Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical ...challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.
DENN Domain Proteins: Regulators of Rab GTPases Marat, Andrea L.; Dokainish, Hatem; McPherson, Peter S.
Journal of biological chemistry/The Journal of biological chemistry,
04/2011, Letnik:
286, Številka:
16
Journal Article
Recenzirano
Odprti dostop
The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies ...reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.
The members of the Rab family of GTPases are master regulators of cellular membrane trafficking. With ∼70 members in humans, Rabs have been implicated in all steps of membrane trafficking ranging ...from vesicle formation and transport to vesicle docking/tethering and fusion. Vesicle trafficking controls the localization and levels of a myriad of proteins, thus regulating cellular functions including proliferation, metabolism, cell-cell adhesion, and cell migration. It is therefore not surprising that impairment of Rab pathways is associated with diseases including cancer. In this review, we highlight evidence supporting the role of Rab13 as a potent driver of cancer progression.
Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a fatal brain disorder featuring cerebellar neurodegeneration leading to spasticity and ataxia. This disease is caused by mutations in the ...SACS gene that encodes sacsin, a massive 4579-amino acid protein with multiple modular domains. However, molecular details of the function of sacsin are not clear. Here, using live cell imaging and biochemistry, we demonstrate that sacsin binds to microtubules and regulates microtubule dynamics. Loss of sacsin function in various cell types, including knockdown and KO primary neurons and patient fibroblasts, leads to alterations in lysosomal transport, positioning, function, and reformation following autophagy. Each of these phenotypic changes is consistent with altered microtubule dynamics. We further show the effects of sacsin are mediated at least in part through interactions with JIP3, an adapter for microtubule motors. These data reveal a new function for sacsin that explains its previously reported roles and phenotypes.
Unc‐51‐like kinases (ULKs) are the most upstream kinases in the initiation of autophagy, yet the molecular mechanisms underlying their function are poorly understood. We report a new role for ULK in ...the induction of autophagy. ULK‐mediated phosphorylation of the guanine nucleotide exchange factor DENND3 at serines 554 and 572 upregulates its GEF activity toward the small GTPase Rab12. Through binding to LC3 and associating with LC3‐positive autophagosomes, active Rab12 facilitates autophagosome trafficking, thus establishing a crucial role for the ULK/DENND3/Rab12 axis in starvation‐induced autophagy.
Synopsis
The GEF DENND3 is shown to have a crucial role in autophagy, under the control of ULK‐mediated phosphoregulation. Phosphorylation upregulates DENND3 GEF activity towards Rab12, facilitating autophagosome trafficking.
This study reveals regulation of membrane trafficking downstream of a key signal transduction pathway, uncovering a new mechanism of ULK function in autophagy.
We provide the first evidence that ULK phosphorylation of a substrate creates a 14‐3‐3 docking site.
Rab12 binds LC3 and co‐localizes with autophagosomes, facilitating autophagosome trafficking
The GEF DENND3 is shown to have a crucial role in autophagy, under the control of ULK‐mediated phosphoregulation. Phosphorylation upregulates DENND3 GEF activity towards Rab12, facilitating autophagosome trafficking.
Neurodegenerative diseases are amongst the most devastating of human disorders. New technologies have led to a rapid increase in the identification of disease-related genes with an enhanced ...appreciation of the key roles played by genetics in the etiology of these disorders. Importantly, pinpointing the normal function of disease gene proteins leads to new understanding of the cellular machineries and pathways that are altered in the disease process. One such emerging pathway is membrane trafficking in the endosomal system. This key cellular process controls the localization and levels of a myriad of proteins and is thus critical for normal cell function. In this review we will focus on three neurodegenerative diseases; Parkinson disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegias, for which a large number of newly discovered disease genes encode proteins that function in endosomal membrane trafficking. We will describe how alterations in these proteins affect endosomal function and speculate on the contributions of these disruptions to disease pathophysiology.
Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and ...signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.
Rab35: GEFs, GAPs and Effectors Chaineau, Mathilde; Ioannou, Maria S.; McPherson, Peter S.
Traffic,
November 2013, Letnik:
14, Številka:
11
Journal Article
Recenzirano
Odprti dostop
Rabs are the largest family of small GTPases and are master regulators of membrane trafficking. Following activation by guanine‐nucleotide exchange factors (GEFs), each Rab binds a specific set of ...effector proteins that mediate the various downstream functions of that Rab. Then, with the help of GTPase‐activating proteins, the Rab converts GTP to GDP, terminating its function. There are over 60 Rabs in humans and only a subset has been analyzed in any detail. Recently, Rab35 has emerged as a key regulator of cargo recycling at endosomes, with an additional role in regulation of the actin cytoskeleton. Here, we will focus on the regulation of Rab35 activity by the connecdenn/DENND1 family of GEFs and the TBC1D10/EPI64 family of GTPase‐activating proteins. We will describe how analysis of these proteins, as well as a plethora of Rab35 effectors has provided insights into Rab35 function. Finally, we will describe how Rab35 provides a novel link between the Rab and Arf family of GTPases with implications for tumor formation and invasiveness.
The small GTPases Rab35 has emerged as a key regulator of endosomal membrane trafficking and actin cytoskeletal dynamics. Here we describe the GEFs that activate Rab35, the GAPs that inactivate Rab35, and the effectors that mediate its diverse biological functions.
Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function can cause disease, including neurodegenerative disorders. The spatiotemporal distribution of lysosomes ...is regulated by small GTPases including Rabs, which are activated by guanine nucleotide exchange factors (GEFs). DENN domain proteins are the largest family of Rab GEFs. Using a cell-based assay, we screened DENND6A, a member of the DENN domain protein family against all known Rabs and identified it as a potential GEF for 20 Rabs, including Rab34. Here, we demonstrate that DENND6A activates Rab34, which recruits a RILP/dynein complex to lysosomes, promoting lysosome retrograde transport. Further, we identify DENND6A as an effector of Arl8b, a major regulatory GTPase on lysosomes. We demonstrate that Arl8b recruits DENND6A to peripheral lysosomes to activate Rab34 and initiate retrograde transport, regulating nutrient-dependent lysosomal juxtanuclear repositioning. Loss of DENND6A impairs autophagic flux. Our findings support a model whereby Arl8b/DENND6A/Rab34-dependent lysosomal retrograde trafficking controls autophagy.
Mechanisms of local protein quality control in the presynaptic nerve terminal remain largely unknown. In this issue of Neuron, Uytterhoeven et al. (2015) describe a form of synaptic endosomal ...microautophagy controlling protein rejuvenation, critical for optimal synaptic activity.
Mechanisms of local protein quality control in the presynaptic nerve terminal remain largely unknown. In this issue of Neuron, Uytterhoeven et al. describe a form of synaptic endosomal microautophagy controlling protein rejuvenation, critical for optimal synaptic activity.
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