Newly endocytosed integral cell surface proteins are typically either directed for degradation or subjected to recycling back to the plasma membrane. The sorting of integral cell surface proteins, ...including signalling receptors, nutrient transporters, ion channels, adhesion molecules and polarity markers, within the endolysosomal network for recycling is increasingly recognized as an essential feature in regulating the complexities of physiology at the cell, tissue and organism levels. Historically, endocytic recycling has been regarded as a relatively passive process, where the majority of internalized integral proteins are recycled via a nonspecific sequence-independent 'bulk membrane flow' pathway. Recent work has increasingly challenged this view. The discovery of sequence-specific sorting motifs and the identification of cargo adaptors and associated coat complexes have begun to uncover the highly orchestrated nature of endosomal cargo recycling, thereby providing new insight into the function and (patho)physiology of this process.
Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the ...plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission.
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•Dynamin is organized as a 2-start helix on membranes primed for fission•Dynamin-decorated lipid tubules achieve super-constriction through GTP hydrolysis•Cryo-EM captures PH domain variable loops 1 and 4 interacting with membranes•Cryo-ET shows dynamin assemblies in vivo as an assortment of helical polymers
Dynamin severs endocytic vesicles. Jimah and Kundu et al. discovered that dynamin, in a post-GTP-hydrolysis state, organizes into a super-constricted 2-start helix on membranes, ready for spontaneous membrane fission. Cryo-ET of dynamin assemblies in vivo revealed that they can accommodate a range of diameters, enabling membrane constriction and fission.
The blood–brain barrier (BBB) is an evolutionarily conserved, structural, and functional separation between circulating blood and the central nervous system (CNS). By controlling permeability into ...and out of the nervous system, the BBB has a critical role in the precise regulation of neural processes. Here, we review recent studies demonstrating that permeability at the BBB is dynamically controlled by circadian rhythms and sleep. An endogenous circadian rhythm in the BBB controls transporter function, regulating permeability across the BBB. In addition, sleep promotes the clearance of metabolites along the BBB, as well as endocytosis across the BBB. Finally, we highlight the implications of this regulation for diseases, including epilepsy.
Recent evidence from fly models reveals an endogenous circadian rhythm at the BBB. This rhythm controls function of the permeability-glycoprotein multidrug transporter, which actively pumps both endogenous and exogenous molecules out of the CNS. Function of this transporter is decreased at night, increasing permeability to multiple substrates into the brain overnight.Endocytosis is a newly appreciated important function for sleep. Endocytosis across the BBB is increased during sleep and, when inhibited, increases the need for sleep.Sleep also promotes the clearance of metabolites out of the brain along paravascular spaces. During sleep, the interstitial spaces of the brain are larger, allowing for more robust movement of metabolites from interstitial spaces into the CSF. These solutes can then travel along vessels and out of the brain.
Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-β peptide (Aβ) is associated with AD. However, ...identifying new compounds that antagonize the underlying cellular pathologies caused by Aβ has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Aβ toxicity where the Aβ peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aβ 1—42 peptide forms more oligomers and is more toxic than Aβ 1—40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of ∼140,000 compounds for rescue of Aβ toxicity. Of ∼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aβ burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aβ secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aβ peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Aβ oligomer formation contributes to Aβ toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aβ yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.
Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root ...cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in m subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.
Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins ...to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin-mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.
Phosphatidylserine (PS), a negatively charged phospholipid present predominantly at the inner leaflet of the plasma membrane, has been widely implicated in many cellular processes including membrane ...trafficking. Along this line, PS has been demonstrated to be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin‐mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell‐attached capacitance measurements that offer millisecond time resolution, we demonstrate in the present study that the fission‐pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission during CME. Furthermore, our results show that the PS‐mediated effect on the fission‐pore duration is Ca2+‐dependent and abolished in the absence of synaptotagmin 1 (Syt1), implying that Syt1 is necessary for the stimulatory role of PS in vesicle fission during CME. Consistently, a Syt1 mutant with a defective PS–Syt1 interaction increases the fission‐pore duration. Taken together, our study suggests that PS–Syt1 interaction may be critical in regulating fission dynamics during CME.
Phosphatidylserine (PS), a negatively charged phospholipid, may be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin‐mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell‐attached capacitance recordings, we demonstrate that the fission‐pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission. Furthermore, the PS‐mediated effect is abolished in the absence of synaptotagmin 1 (Syt1) and a Syt1 mutant with a defective PS–Syt1 interaction increases the fission‐pore duration. Our study thus suggests an importance of the PS–Syt1 interaction in regulating fission dynamics during CME.
Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits ...against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.
The discovery of genetic forms of Parkinson’s disease (PD) has highlighted the importance of the autophagy/lysosomal and mitochondrial/oxidative stress pathways in disease pathogenesis. However, ...recently identified PD-linked genes, including DNAJC6 (auxilin), SYNJ1 (synaptojanin 1), and the PD risk gene SH3GL2 (endophilin A1), have also highlighted disruptions in synaptic vesicle endocytosis (SVE) as a significant contributor to disease pathogenesis. Additionally, the roles of other PD genes such as LRRK2, PRKN, and VPS35 in the regulation of SVE are beginning to emerge. Here we discuss the recent work on the contribution of dysfunctional SVE to midbrain dopaminergic neurons’ selective vulnerability and highlight pathways that demonstrate the interplay of synaptic, mitochondrial, and lysosomal dysfunction in the pathogenesis of PD.
Emerging genetic and mechanistic studies link synaptic, mitochondrial, and lysosomal dysfunction as major contributors to the degeneration of midbrain dopaminergic neurons.
Recently, endocytic genes (DNAJC6, SYNJ1, and SH3GL2) have been linked to Parkinson’s disease pathogenesis or identified as a risk factor for the disease, implicating a role for impaired synaptic vesicle endocytosis (SVE) in neurodegeneration.
Among the modifiers of proteins involved in SVE are LRRK2 and parkin. LRRK2’s and parkin’s involvement in this context is through their phosphorylation and ubiquitination actions, respectively.
Dysfunction in SVE in dopaminergic neurons can lead to increased levels of unpackaged, cytosolic DA that is subject to oxidation and pathogenic downstream effects.
Clathrin-mediated endocytosis (CME) regulates signaling from the plasma membrane. Analysis of clathrin-coated pit (CCP) dynamics led us to propose the existence of a rate-limiting, regulatory step(s) ...that monitor the fidelity of early stages in CCP maturation. Here we show that nascent endocytic vesicles formed in mutant cells displaying rapid, dysregulated CME are defective in early endosomal trafficking, maturation and acidification, confirming the importance of this "checkpoint." Dysregulated CME also alters EGF receptor signaling and leads to constitutive activation of the protein kinase Akt. Dynamin-1, which was thought to be neuron specific, is activated by the Akt/GSK3beta signaling cascade in non-neuronal cells to trigger rapid, dysregulated CME. Acute activation of dynamin-1 in RPE cells by inhibition of GSK3beta accelerates CME, alters CCP dynamics and, unexpectedly, increases the rate of CCP initiation. CRISPR-Cas9n-mediated knockout and reconstitution studies establish that dynamin-1 is activated by Akt/GSK3beta signaling in H1299 non-small lung cancer cells. These findings provide direct evidence for an isoform-specific role for dynamin in regulating CME and reveal a feed-forward pathway that could link signaling from cell surface receptors to the regulation of CME. Synopsis Dynamin-1, previously thought to be neuron specific, is activated by an Akt/GSK3beta signaling cascade in non-neuronal cells. Dynamin-1 activation alters the rate and regulation of clathrin-mediated endocytosis, providing a feed-forward pathway between endocytosis and signaling. An endocytic checkpoint monitors the fidelity of clathrin-coated vesicle (CCV) formation. Dysregulated clathrin-mediated endocytosis (CME) alters cell signaling and proliferation. Dynamin-1 and dynamin-2 differentially regulate early stages of CME. Endogenous dynamin-1 is activated by an Akt/GSK3beta signaling cascade, illustrating a first non-neuronal role of dynamin-1.
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