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Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

Alphaherpesvirus tegument assembly, secondary envelopment, and exocytosis processes are understood in broad strokes, but many of the individual steps in this pathway, and their molecular and cell biological details, remain unclear. Viral tegument and membrane proteins form an extensive and robust protein interaction network, such that essentially any structural protein can be deleted, yet particles are still assembled, enveloped, and released from infected cells. We conceptually divide the tegument proteins into three groups: conserved inner and outer teguments that participate in nucleocapsid and membrane contacts, respectively, and 'middle' tegument proteins, consisting of some of the most abundant tegument proteins that serve as central hubs in the protein interaction network, yet which are unique to the alphaherpesviruses. We then discuss secondary envelopment, reviewing the tegument-membrane contacts and cellular factors that drive this process. We place this viral process in the context of cell biological processes, including the endocytic pathway, ESCRT machinery, autophagy, secretory pathway, intracellular transport, and exocytosis mechanisms. Finally, we speculate about potential relationships between cellular defenses against oligomerizing or aggregating membrane proteins and the envelopment and egress of viruses.

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel β subunit (CaVβ), and the binding between CaVα1/CaVβ appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVβ. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVβ independent promotion of dynamin-dependent endocytosis.

‘Candidatus Liberibacter asiaticus’ (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas-efficient propagation in the vector, the Asian citrus psyllid Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut, the major organ for CLas transmission, is fundamental to improving current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles, termed Liberibacter-containing vacuoles (LCVs), by endocytosis. Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). Additionally, we observed the formation of double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas uses endocytosis- and exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic, and exocytic pathways via complex machineries. Potential tactics for HLB control can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.

Late endosome and lysosomes form the second major membrane network along the endocytic pathway, after early/recycling endosomes, and as such, late endosomes function as the last portal before lysosomes. In mammalian cells, they also link the endosomal system to other cellular destinations, including the trans-Golgi complex and the plasma membrane. Finally it has become clear in recent years that late endosomes operate as a major sensing and signaling organelle that informs the cell about its nutrient and supply situation.

Early endosomes (EEs) are part of the endocytic transport pathway and resemble the earliest class of transport vesicles between the internalization of extracellular material, their cellular distribution or vacuolar degradation. In filamentous fungi, EEs fulfill important functions in long distance transport of cargoes as mRNAs, ribosomes, and peroxisomes. Formation and maturation of early endosomes is controlled by the specific membrane-bound Rab-GTPase Rab5 and tethering complexes as CORVET (class C core vacuole/endosome tethering). In the basidiomycete Ustilago maydis, Rab5a is the prominent GTPase to recruit CORVET to EEs; in rab5a deletion strains, this function is maintained by the second EE-associated GTPase Rab5b. The tethering- and core-subunits of CORVET are essential, buttressing a central role for EE transport in U. maydis. The function of EEs in long distance transport is supported by the Nma1 protein that interacts with the Vps3 subunit of CORVET. The interaction stabilizes the binding of Vps3 to the CORVET core complex that is recruited to Rab5a via Vps8. Deletion of nma1 leads to a significantly reduced number of EEs, and an increased conversion rate of EEs to late endosomes. Thus, Nma1 modulates the lifespan of EEs to ensure their availability for the various long distance transport processes.

During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTB(fl/fl) mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.

Like most eukaryotic organisms, fungi use endocytosis for nutrition, signal transduction, turnover of plasma membrane molecules, etc. It is generally accepted that in filamentous fungi, as in yeast, invaginations of the plasma membrane of a small size (up to about 100 nm) are formed in the early stages of endocytosis. These invaginations are surrounded by a rigid actin scaffold – an actin patch, and give rise to small primary endocytic vesicles after scission from the plasma membrane. However, in classical mycological studies, complex large-volume invaginations of the plasma membrane – lomasomes – were described in filamentous fungi. In our time, in a number of filamentous basidiomycetes when tracking endocytosis using styryl fluorescent labels, large invaginations of the plasma membrane have been found, presumably forming endocytic macrovesicles after scission. In this paper, for comparison, large-sized types of endocytosis in animal cells are briefly described. Information about tubular endocytic invaginations in fungi is presented. Three types of large invaginations of the plasma membrane, detected at the TEM level in basidiomycetes, are characterized. The main question this paper addresses is whether or not filamentous fungi do have an analogue of animal macropinocytosis – macrovesicular endocytosis. There are some indications that the answer to this question is yes, but further research is needed. The presence of macrovesicular endocytosis may change the well-established beliefs about the cellular organization of filamentous fungi and the physiology of their nutrition.

Hedgehog proteins (Hhs) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholesteroylation attach Hhs to membranes, and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that polarized Madin–Darby canine kidney cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholesteroylation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is secreted unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from Rab11-ARE. At the steady state, Shh predominates apically and can be basolaterally transcytosed. This Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.

Communication between organelles is an essential process that helps maintain cellular homeostasis and organelle contact sites have recently emerged as crucial mediators of this communication. The emergence of a class of molecular bridges that span the inter-organelle gaps has now been shown to direct the flow of lipid traffic from one lipid bilayer to another. One of the key components of these molecular bridges is the presence of an N-terminal Chorein/VPS13 domain. This is an evolutionarily conserved domain present in multiple proteins within the endocytic and autophagy trafficking pathways. Herein, we discuss the current state-of-the-art of this class of proteins, focusing on the role of these lipid transporters in the autophagy and endocytic pathways. We discuss the recent biochemical and structural advances that have highlighted the essential role Chorein-N domain containing ATG2 proteins play in driving the formation of the autophagosome and how lipids are transported from the endoplasmic reticulum to the growing phagophore. We also consider the VPS13 proteins, their role in organelle contacts and the endocytic pathway and highlight how disease-causing mutations disrupt these contact sites. Finally, we open the door to discuss other Chorein_N domain containing proteins, for instance, UHRF1BP1/1L, their role in disease and look towards prokaryote examples of Chorein_N-like domains. Taken together, recent advances have highlighted an exciting opportunity to delve deeper into inter-organelle communication and understand how lipids are transported between membrane bilayers and how this process is disrupted in multiple diseases.

EGFR plays key roles in multiple cellular processes such as cell differentiation, cell proliferation, migration and epithelia homeostasis. Phosphorylation of the receptor, intracellular signaling and trafficking are major events regulating EGFR functions. Galectin-7, a soluble lectin expressed in epithelia such as the skin, has been shown to be involved in cell differentiation. Through this study we demonstrate that galectin-7 regulates EGFR function by a direct interaction with its extracellular domain hence modifying its downstream signaling and endocytic pathway. From observations in mice we focused on the molecular mechanisms deciphering the glycosylation dependent interaction between EGFR and galectin-7. Interestingly, we also revealed that galectin-7 is a direct binder of both EGFR and E-cadherin bridging them together. Strikingly this study not only deciphers a new molecular mechanism of EGFR regulation but also points out a novel molecular interaction between EGFR and E-cadherin, two major regulators of the balance between proliferation and differentiation.SUMMARYEGFR and E-cadherin are known to interact and to regulate epithelial homeostasis. In this study we unravel in the epidermis a new partner and regulator of EGFR which also binds E-cadherin reciprocally bridging their dynamics and functions.

The ligand-induced internalization of epidermal growth factor receptor (EGFR) is generally considered to attenuate downstream signaling via its endosomal degradation. However, the endocytosis of an oncogenic EGFR variant III (EGFRvIII) is impaired, which leads to persistent signaling from the cell surface, thereby promoting the proliferation and survival of glioblastoma multiforme (GBM) cells. Cellular stress triggers the non-canonical endocytosis-recycling of EGFR by p38-mediated phosphorylation. In the present study, we used temozolomide (TMZ), the standard chemotherapeutic agent for the treatment of GBM patients, to examine whether EGFRvIII is controlled by a non-canonical mechanism. TMZ triggered the endocytic trafficking of serine phosphorylated EGFRvIII. Moreover, phosphorylation and endocytosis were abrogated by the selective p38 inhibitor SB203580, but not gefitinib, indicating that EGFRvIII is recruited to p38-mediated non-canonical endocytosis. The combination of TMZ and SB203580 also showed potential inhibitory effects on the proliferation and motility of glioblastoma cells.

Brain-derived neurotrophic factor (BDNF) regulates diverse brain functions via TrkB receptor signaling. Due to the expression of TrkB receptors, astrocytes can internalize extracellular BDNF proteins via receptor-mediated endocytosis. Endocytosed BDNF can be re-secreted upon stimulation, but the molecular mechanism underlying this phenomenon remains unrecognized. Our study reveals that vesicle-associated membrane protein 3 (Vamp3) selectively regulates the release of endocytic BDNF from astrocytes. By using quantum dot (QD)-conjugated mature BDNF (QD-BDNF) as a proxy for the extracellular BDNF protein, we monitored the uptake, transport, and secretion of BDNF from cultured cortical astrocytes. Our data showed that endocytic QD-BDNF particles were enriched in Vamp3-containing vesicles in astrocytes and that ATP treatment sufficiently triggered either the antero- or retrograde transport and exocytosis of QD-BDNF-containing vesicles. Downregulation of Vamp3 expression disrupted endocytic BDNF secretion from astrocytes but did not affect uptake or transport. Collectively, these results provide evidence of the selective ability of astrocytic Vamp3 to control endocytic BDNF secretion during BDNF recycling.

Ligand-independent EGFR endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid signaling towards the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and PKA activity. This PA/PDE4/PKA pathway induces clathrin-dependent and clathrin-independent endocytosis followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this PA/PDE4/PKA pathway using biosensors of PA, cAMP and PKA in living cells. Propranolol used to inhibit PA hydrolysis triggers this pathway, which then activates p38 and ERK1/2 downstream PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increased the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is likely due to EGFR phosphorylation, as detected in Thr1047/1047 and Ser669 residues. However, EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin and Rab11, while a small proportion distributes to Alix-endosomes. This indicates a recycling arrest, which includes includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer. This article is protected by copyright. All rights reserved.

Endocytosis is an essential mechanism for cellular uptake in many human tissues. A range of endocytic mechanisms occur including clathrin-dependent and -independent mechanisms. However, the role of endocytosis in the placenta and the spatial localisation of individual mechanisms is not well understood. The two principal cell layers that comprise the placental barrier to maternal-fetal transfer are the syncytiotrophoblast and fetal capillary endothelium. Endocytic uptake into the syncytiotrophoblast has been demonstrated for physiological maternal molecules such as transferrin-bound iron and low density lipoprotein (LDL) and may play an important role in the uptake of several other micronutrients, serum proteins, and therapeutics at both major placental cell barriers. These mechanisms may also mediate placental uptake of some viruses and nanoparticles. This review introduces the mechanisms of cargo-specific endocytosis and what is known about their localisation in the placenta, focussing predominantly on the syncytiotrophoblast. A fuller understanding of placental endocytosis is necessary to explain both fetal nutrition and the properties of the placental barrier. Characterising placental endocytic mechanisms and their regulation may allow us to identify their role in pregnancy pathologies and provide new avenues for therapeutic intervention.

Membrane contact sites (MCSs) refer to the areas of close proximity between heterologous membranes. A growing body of evidence indicates that MCSs are involved in important cellular functions, such as cellular material transfer, organelle biogenesis, and cell growth. Importantly, the study of MCSs at the bacteria-host interface is an emerging popular research topic. Intracellular bacterial pathogens have evolved a variety of fascinating strategies to interfere with MCSs by injecting effectors into infected host cells. Bacteria-containing vacuoles establish direct physical contact with organelles within the host, ensuring vacuolar membrane integrity and energy supply from host organelles and protecting the vacuoles from the host endocytic pathway and lysosomal degradation. An increasing number of bacterial effectors from various bacterial pathogens hijack components of host MCSs to form the vacuole-organelle MCSs for material exchange. MCS-related events have been identified as new mechanisms of microbial pathogenesis to greatly improve bacterial survival and replication within host cells. In this review, we will discuss the recent advances in MCSs at the bacteria-host interface, focussing on the roles of MCSs mediated by bacterial effectors in microbial pathogenesis.

TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its overexpression being associated with familial frontotemporal lobar degeneration, and point mutation linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV-2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a nonstandard way to show that the lumenal portion of TMEM106B is a member of the late embryogenesis abundant-2 (LEA-2) domain superfamily. More sensitive tools (HMMER, HHpred, and trRosetta) extended this to predict LEA-2 domains in two yeast proteins. One is Vac7, a regulator of PI(3,5)P2 production in the degradative vacuole, equivalent to the lysosome, which has a LEA-2 domain in its lumenal domain. The other is Tag1, another vacuolar protein, which signals to terminate autophagy and has three LEA-2 domains in its lumenal domain. Further analysis of LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid-binding groove. This implies that TMEM106B, Vac7, and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles.

Specificity and sensitivity in biochemical reactions can be achieved through regulation of equilibrium binding affinity or through proofreading mechanisms that allow for the dissociation of unwanted intermediates. In this essay, we aim to provide our perspectives on how the concept of kinetic proofreading might apply in the context of cargo sorting in clathrin-mediated endocytosis.

Endocytic traffic is a complex and elegant operation involving cargo sorting, membrane budding and tubulation, generation of force, and the formation of organellar contacts. The role of specific proteins and lipids in these processes has been studied extensively. By comparison, precious little is understood about the contribution of the endocytic fluid to these events, despite much evidence that alteration of the contents can severely affect membrane traffic along the endocytic pathway. In particular, it has long been appreciated that dissipation of ionic gradients arrests endosome-to-lysosome maturation. How cells sense inorganic ions and transmit this information have remained largely enigmatic. Herein, we review the experimental findings that reveal an intimate association between luminal ions, their transport, and endocytic traffic. We then discuss the ionic sensors and the mechanisms proposed to convert ion concentrations into protein-based trafficking events, highlighting the current paucity of convincing explanations.

Phosphorothioate-modified antisense oligonucleotide (PS-ASO) drugs are commonly used to modulate gene expression through RNase H1-mediated cleavage of target RNAs. Upon internalization through endocytic pathways into cells, PS-ASOs must be released from membraned endosomal organelles to act on target RNAs, a limiting step of PS-ASO activity. Here we report that Hsc70 protein mediates productive release of PS-ASOs from endosomes. Hsc70 protein was enriched in endosome fractions shortly after PS-ASO incubation with cells. Reduction of Hsc70 significantly decreased the activities of PS-ASOs in reducing target RNAs. PS-ASO uptake and transport from early endosomes to late endosomes (LEs) were not affected upon Hsc70 reduction; however, endosomal release of PS-ASOs was impaired. Reduction of Hsc70 led to more scattered mannose-6-phosphate receptor (M6PR) localization at LEs in the cytoplasm, in contrast to the perinuclear localization at trans-Golgi network (TGN) in control cells, suggesting that retrograde transport of M6PR from LEs to TGN was affected. Consistently, reduction of Hsc70 increased colocalization of M6PR and PS-ASOs at LEs, and also delayed M6PR antibody transport from LE to TGN. Together, these results suggest that Hsc70 protein is involved in M6PR vesicle escape from LEs and may thus enhance PS-ASO release from LEs.

Endocytosis mediates the uptake of extracellular proteins, micronutrients and transmembrane cell surface proteins. Importantly, many viruses, toxins and bacteria hijack endocytosis to infect cells. The canonical pathway is clathrin-mediated endocytosis (CME) and is active in all eukaryotic cells to support critical house-keeping functions. Unconventional mechanisms of endocytosis exit in parallel of CME, to internalize specific cargoes and support various cellular functions. These clathrin-independent endocytic (CIE) routes use three distinct mechanisms: acute signaling-induced membrane remodeling drives macropinocytosis, activity-dependent bulk endocytosis (ADBE), massive endocytosis (MEND) and EGFR non-clathrin endocytosis (EGFR-NCE). Cargo capture and local membrane deformation by cytosolic proteins is used by fast endophilin-mediated endocytosis (FEME), IL-2Rβ endocytosis and ultrafast endocytosis at synapses. Finally, the formation of endocytic pits by clustering of extracellular lipids or cargoes according to the Glycolipid-Lectin (GL-Lect) hypothesis mediates the uptake of SV40 virus, Shiga and cholera toxins, and galectin-clustered receptors by the CLIC/GEEC and the endophilin-A3-mediated CIE.

Caveolae are abundant plasma membrane pits formed by the coordinated action of peripheral and integral membrane proteins and membrane lipids. Here, we discuss recent studies that are starting to provide a glimpse of how filamentous cavin proteins, membrane-embedded caveolin proteins, and specific plasma membrane lipids are brought together to make the unique caveola surface domain. Protein assembly involves multiple low-affinity interactions that are dependent on 'fuzzy' charge-dependent interactions mediated in part by disordered cavin and caveolin domains. We propose that cavins help generate a lipid domain conducive to full insertion of caveolin into the bilayer to promote caveola formation. The synergistic assembly of these dynamic protein complexes supports the formation of a metastable membrane domain that can be readily disassembled both in response to cellular stress and during endocytic trafficking. We present a mechanistic model for generation of caveolae based on these new insights.

The trafficking of G protein–coupled receptors (GPCRs) to different membrane compartments has recently emerged as being a critical determinant of the signaling profiles of activation. GPCRs, which share many structural and functional similarities, also share many mechanisms that traffic them between compartments. This sharing raises the question of how the trafficking of individual GPCRs is selectively regulated. Here, we will discuss recent studies addressing the mechanisms that contribute to selectivity in endocytic and biosynthetic trafficking of GPCRs.

For the protist parasite Entamoeba histolytica, endocytic processes, such as phagocytosis, are essential for its survival in the human gut. The actin cytoskeleton is involved in the formation of pseudopods and phagosomal vesicles by incorporating a number of actin-binding and modulating proteins along with actin in a temporal manner. The actin dynamics, which comprises polymerization, branching, and depolymerization is very tightly regulated and takes place directionally at the sites of initiation of phagocytosis. Formin and profilin are two actin-binding proteins that are known to regulate actin cytoskeleton dynamics and thereby, endocytic processes. In this article, we report the participation of formin and profilin in E. histolytica phagocytosis and propose that these two proteins interact with each other and their sequential recruitment at the site is required for the successful completion of phagocytosis. The evidence is based on detailed microscopic, live imaging, interaction studies, and expression downregulation. The cells downregulated for expression of formin show absence of profilin at the site of phagocytosis, whereas downregulation of profilin does not affect formin localization.

Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temperature, pH or free radical generation). HeLa cells are interesting to study with these nanodiamonds since they are a model cell system that is widely used to study cancer-related diseases. However, they only internalize low numbers of nanodiamond particles very slowly via the endocytosis pathway. In this work, we show that pH-sensitive, dextran-coated fluorescent nanodiamonds can be used to visualise this pathway. Additionally, this coating improved diamond uptake in HeLa cells by 5.3 times (*** p &lt, 0.0001) and decreased the required time for uptake to only 30 min. We demonstrated further that nanodiamonds enter HeLa cells via endolysosomes and are eventually expelled by cells.

Clathrin-dependent endocytosis and exocytosis are coordinated to maintain cell homeostasis in Arabidopsis roots., Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.

The plasma-membrane marker FM1-43 was employed to reveal the relative significance of different types of endocytic and transcytic mechanisms in outer hair cells (OHCs) of the guinea-pig cochlea. A double-barrel local perfusion system was used to label independently the apical or synaptic pole of the isolated OHC, to study mechanisms of vesicle uptake at the poles and of vesicle trafficking along and across the cell. Treatment with an inhibitor of macropino- and phagocytosis, phenylarsine oxide, or of clathrin-mediated endocytic activity, concanavalin A, significantly reduced the dye uptake at both the apical and the synaptic poles, indicating the presence of both clathrin-independent and clathrin-mediated processes at both poles. However, measurement of uptake speed in the presence of the inhibitors suggested that clathrin-independent processes contribute more extensively to endocytosis at the basal pole than the apical pole. Treatment with an inhibitor of myosin VI, 2,4,6-triiodophenol, significantly delayed both the apicobasal and the basoapical fluorescence signals. However, treatment with an inhibitor of kinesin, monastrol, or of dynein, ciliobrevin D, significantly delayed the signals only in the basoapical direction. The myosin-VI inhibitor, but neither the kinesin nor dynein inhibitors, significantly delayed the signals to the subsurface cisternae. That is, myosin VI carries vesicles in both longitudinal directions as well as radially to the sub-surface cisternae, whereas kinesin and dynein participate primarily in basoapical trafficking. This fundamental information is essential for elucidating recycling mechanisms of specific proteins involved in establishing, controlling, and maintaining the electromechanical action of OHCs and, therefore, vital for understanding auditory perception.

Flaviviruses are enveloped viruses that infect multiple hosts. Envelope proteins are the outermost proteins in the structure of flaviviruses and mediate viral infection. Studies indicate that flaviviruses mainly use envelope proteins to bind to cell attachment receptors and endocytic receptors for the entry step. Here, we present current findings regarding key envelope protein amino acids that participate in the flavivirus early infection process. Among these sites, most are located in special positions of the protein structure, such as the α-helix in the stem region and the hinge region between domains I and II, motifs that potentially affect the interaction between different domains. Some of these sites are located in positions involved in conformational changes in envelope proteins. In summary, we summarize and discuss the key envelope protein residues that affect the entry process of flaviviruses, including the process of their discovery and the mechanisms that affect early infection.

Cells are remarkably resilient structures, they are able to recover from injuries to their plasma membrane (PM) and cytoskeleton that would normally constitute existential threats. This capacity is exemplified by Xenopus laevis oocytes which can recover from very large PM defects through exocytotic and endocytic events and can repair damaged cortical cytoskeleton structures through the formation of a contractile actomyosin ring (AMR). Formation of the AMR involves the localized Ca2+ -dependent activation of RhoA and Cdc42, and the pre-patterning of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, this model fails to account for observations that suggest a link between cytoskeletal dynamics, intracellular tension and AMR formation. It also does not explain why the formation of an AMR is not involved in the cytoskeletal repair program of adherent cells. We show here evidence for the support of tension as an essential regulatory signal for the formation of AMR. Indeed, oocytes in which global tension has been experimentally reduced were unable to form a functional AMR following injury, showing severely diminished RhoA activity at the wound site. These new insights place the cytoskeleton at the center of events involving changes in cell shape such as cytokinesis which also involves the formation and closure of an AMR. This article is protected by copyright. All rights reserved.

Synthetic therapeutic oligonucleotides (STO) represent the third bonafide platform for drug discovery in the pharmaceutical industry after small molecule and protein therapeutics. So far, thirteen STOs have been approved by regulatory agencies and over one hundred of them are in different stages of clinical trials. STOs hybridize to their target RNA or DNA in cells via Watson-Crick base pairing to exert their pharmacological effects. This unique class of therapeutic agents has the potential to target genes and gene products that are considered undruggable by other therapeutic platforms. However, STOs must overcome several extracellular and intracellular obstacles to interact with their biological RNA targets inside cells. These obstacles include degradation by extracellular nucleases, scavenging by the reticuloendothelial system, filtration by the kidney, traversing the capillary endothelium to access the tissue interstitium, cell-surface receptor-mediated endocytic uptake, and escape from endolysosomal compartments to access the nuclear and/or cytoplasmic compartments where their targets reside. In this review, we present the recent advances in this field with a specific focus on antisense oligonucleotides (ASOs) and siRNA therapeutics.

The uptake of receptors by clathrin-mediated endocytosis underlies signaling, nutrient import, and recycling of transmembrane proteins and lipids. In the complex, crowded environment of the plasma membrane, receptors are internalized when they bind to components of the clathrin coat, such as the major adaptor protein, AP2. Receptors with higher affinity for AP2 are known to be more strongly internalized compared to receptors with lower affinity. However, it remains unclear how receptors with different affinities compete for space within crowded endocytic structures. To address this question, we constructed receptors with varying affinities for AP2 and allowed them to compete against one another during internalization. As expected, the internalization of a receptor with high affinity for AP2 was reduced when it was coexpressed with a competing receptor of similar affinity. However, receptors of low affinity for AP2 were surprisingly difficult to displace from endocytic structures, even when expressed alongside receptors with much higher affinity. To understand how these low-affinity receptors are protected from competition, we looked at AP2 heterogeneity across clathrin-coated structures. When we examined structures with lower-than-average AP2 content, we found that they were relatively enriched in cargo of low affinity for AP2 and depleted of cargo with high affinity. These findings suggest that the heterogeneity of adaptor protein content across the population of endocytic structures enables the internalization of diverse receptors. Given the critical role that internalization plays in signaling, this effect may help to prevent strongly internalized receptors from interfering with the cell's ability to process signals from weakly internalized receptors.

CLN5 is a soluble endolysosomal protein whose function is poorly understood. Mutations in this protein cause a rare neurodegenerative disease, Neuronal Ceroid Lipofuscinosis. We previously found that depletion of CLN5 leads to dysfunctional retromer, resulting in the degradation of the lysosomal sorting receptor, sortilin. However, how a soluble lysosomal protein can modulate the function of a cytosolic protein, retromer, is not known. In this work, we show that deletion of CLN5 not only results in retromer dysfunction, but also in impaired endolysosome fusion events. This results in delayed degradation of endocytic proteins and in defective autophagy. CLN5 modulates these various pathways by regulating downstream interactions between CLN3, an endolysosomal integral membrane protein whose mutations also result in Neuronal Ceroid Lipofuscinosis, RAB7A, and a subset of RAB7A effectors. Our data supports a model where CLN3 and CLN5 function as an endolysosomal complex regulating various functions.

Exosomes represent a subtype of extracellular vesicle that is released through retrograde transport and fusion of multivesicular bodies with the plasma membrane1. Although no perfect methodologies currently exist for the high-throughput, unbiased isolation of pure plasma exosomes2,3, investigation of exosome-enriched plasma fractions of extracellular vesicles can confer a glimpse into the endocytic pathway on a systems level. Here we conduct high-coverage lipidomics with an emphasis on sterols and oxysterols, and proteomic analyses of exosome-enriched extracellular vesicles (EVs hereafter) from patients at different temporal stages of COVID-19, including the presymptomatic, hyperinflammatory, resolution and convalescent phases. Our study highlights dysregulated raft lipid metabolism that underlies changes in EV lipid membrane anisotropy that alter the exosomal localization of presenilin-1 (PS-1) in the hyperinflammatory phase. We also show in vitro that EVs from different temporal phases trigger distinct metabolic and transcriptional responses in recipient cells, including in alveolar epithelial cells, which denote the primary site of infection, and liver hepatocytes, which represent a distal secondary site. In comparison to the hyperinflammatory phase, EVs from the resolution phase induce opposing effects on eukaryotic translation and Notch signalling. Our results provide insights into cellular lipid metabolism and inter-tissue crosstalk at different stages of COVID-19 and are a resource to increase our understanding of metabolic dysregulation in COVID-19.

Phosphatidylserine (PS) is involved in various cellular processes in yeast and animals. However, PS functions in plants remain unclear. In Arabidopsis, PS is relatively enriched in flower and root tissues, and the genetic disturbance of PS biosynthesis in phosphatidylserine synthase1 (PSS1)/ pss1 heterozygotes induces sporophytic and gametophytic defects during pollen maturation. This study functionally characterized PS in Arabidopsis roots and observed that pss1 seedlings exhibited a short-root phenotype by reducing the meristem size and cell elongation capacity. Confocal microscopy imaging analyses of PS with GFP-LactC2 and the endocytic activity with FM 4-64 revealed that although GFP-LactC2 (or PS) was localized in the plasma membrane and endocytic membranes, the lack of PS in pss1 roots did not affect the constitutive endocytosis. Instead, a fluorescence imaging analysis of the cytokinetic phases in the dividing zone of pss1-2 roots revealed a significant delay in telophase progression, requiring active cargo vesicle trafficking for cell plate formation. Confocal microscopy imaging analysis of transgenic GFP-LactC2 root cells with developing cell plates indicated that GFP-LactC2 was localized at the cell plate. Moreover, confocal microscopy images of transgenic pss1-2 and PSS1 roots expressing the cell plate-specific syntaxin construct ProKNOLLE:eGFP-KNOLLE showed abnormal cell plate development in pss1-2ProKNOLLE:eGFP-KNOLLE roots. These results suggested that PS is required for root cytokinesis, possibly because it helps mediate the cargo vesicular trafficking required for cell plate formation.

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration–approved pan-ERBB inhibitor employed for the treatment of ERBB2+ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30–100 nm) ERBB2− EVs and large (>100 nm) ERBB2+ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2+ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs

Toxic amyloid-beta (Aβ) peptides, produced by sequential proteolytic cleavage of the amyloid precursor protein (APP), play a key role in the initial stage of Alzheimer's disease (AD). Increasing evidence indicates that Aβ42 induces neuronal circuit hyperexcitability in the early stages of AD pathology. As a result, researchers have investigated treatments that modulate the excitatory/inhibitory imbalance as potential AD therapies. For example, levetiracetam, an atypical antiepileptic drug used to quell hyperexcitability, has garnered recent interest in the AD field, even though its exact mechanism(s) of action remains elusive. Here, we show that in APP knock-in mouse models of amyloid pathology, chronic levetiracetam administration decreases cortical Aβ42 levels and lowers the amyloid plaque burden. In addition, using multiplexed tandem mass tag-quantitative mass spectrometry-based proteomic analysis, we determined that chronic levetiracetam administration selectively normalizes levels of presynaptic endocytic proteins. Finally, we found that levetiracetam treatment selectively lowers beta carboxyl-terminal fragment levels, while the abundance of full-length APP remains unchanged. In summary, this work reports that chronic treatment with levetiracetam serves as a useful therapeutic in AD by normalizing levels of presynaptic endocytic proteins and altering APP cleavage preference, leading to a decrease in both Aβ42 levels and the amyloid plaque burden. These novel findings provide novel evidence for the previously documented therapeutic value of levetiracetam to mitigate AD pathology.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of receptors that bind sialic acids in specific linkages on glycoproteins and glycolipids. Siglecs play roles in immune signalling and exhibit cell-type specific expression and endocytic properties. Recent studies suggest that Siglecs are likely to function as immune checkpoints that regulate responses in cancers and inflammatory diseases. In this review, we discuss strategies to target the Siglec-sialic acid axis in human diseases, particularly cancer, and the possibility of exploiting them for therapeutic intervention.

Serum transferrin (Tf) is the essential iron transport protein in the body. Transferrin is responsible for the sequestration of free iron in serum and the delivery of iron throughout the body and into cells, where iron is released inside a mildly acidified endosome. Altered iron distributions are associated with diseases such as iron-overload, cancer, and cardiovascular disease. The presence of free iron is linked to deleterious redox reactions, inside and outside cells and organelles. As Tf iron release is pH dependent, any changes in intraorganelle and extracellular pH, often associated with disease progression, could inhibit normal iron delivery or accelerate iron release in the wrong compartment. However, imaging approaches to monitor changes in the iron-bound state of Tf are lacking. Recently, Raman spectroscopy has been shown to measure iron-bound forms of Tf in solution, intact cells and tissue samples. Here, a biochemical Raman assay has been developed to identify iron-release from Tf following modification of chemical environment. Quantitative singular value decomposition (SVD) method has been applied to discriminate between iron-bound Tf samples during endocytic trafficking in intact cancer cells subjected to Raman hyperspectral confocal imaging. We demonstrate the strength of the SVD method to monitor pH-induced Tf iron-release using Raman hyperspectral imaging, providing the redox biology field with a novel tool that facilitates subcellular investigation of the iron-binding profile of transferrin in various disease models.

Background/aim Copper metabolism MURR1 domain-containing 5 (COMMD5) is mainly expressed in renal tubules (RTs), where it facilitates re-differentiation of injured RTs. We reported that COMMD5 regulates the expression of epidermal growth factor receptor by participating in its endocytic membrane trafficking, thus inhibiting tumor growth. Here we aimed to determine the role of COMMD5 in malignant phenotypes of renal cell carcinoma (RCC). Materials and methods The associations between COMMD5 levels in RTs adjacent to RCC tumors in patients and their clinicopathologic characteristics were evaluated, and the effects of COMMD5 on cancer stemness in RCC cells were investigated. Results Low COMMD5 levels in RTs correlated with high tumorigenesis and poor patient outcomes. COMMD5 overexpression in RCC cells reduced the proportion of cancer stem cell-like cells and their malignant phenotypes, including proliferation, invasion and sphere formation. Secreted COMMD5 from RT cells also reduced malignant phenotypes. Conclusion COMMD5 might suppress malignant phenotypes of RCC, thus inhibiting tumor development and improving patient prognosis.

Endocytosis allows cells to transport particles and molecules across the plasma membrane. In addition, it is involved in the termination of signalling through receptor downmodulation and degradation. This traditional outlook has been substantially modified in recent years by discoveries that endocytosis and subsequent trafficking routes have a profound impact on the positive regulation and propagation of signals, being key for the spatiotemporal regulation of signal transmission in cells. Accordingly, endocytosis and membrane trafficking regulate virtually every aspect of cell physiology and are frequently subverted in pathological conditions. Two key aspects of endocytic control over signalling are coming into focus: context-dependency and long-range effects. First, endocytic-regulated outputs are not stereotyped but heavily dependent on the cell-specific regulation of endocytic networks. Second, endocytic regulation has an impact not only on individual cells but also on the behaviour of cellular collectives. Herein, we will discuss recent advancements in these areas, highlighting how endocytic trafficking impacts complex cell properties, including cell polarity and collective cell migration, and the relevance of these mechanisms to disease, in particular cancer. The canonical function of endocytosis is molecule internalization. Beyond this role, endocytic trafficking has emerged as a process central to the spatiotemporal regulation of cell signalling. Endocytic trafficking thus controls many cellular processes and tissue-wide properties, including cell migration and polarity, and its deregulation has been implicated in pathologies, particularly cancer.

The precise spatiotemporal control of nanoscale membrane shape and composition is the result of complex interplay of individual and collective molecular behaviors. Here, we employed single-molecule localization microscopy and computational simulations to observe single-lipid diffusion and sorting in model membranes with varying compositions, phase, temperature, and curvature. Supported lipid bilayers were created over 50-nm radius nanoparticles to mimic the size of naturally occurring membrane buds, such as endocytic pits and the formation of viral envelopes. The curved membranes recruited liquid-disordered lipid phases while altering the diffusion and sorting of tracer lipids. Disorder-preferring fluorescent lipids sorted to and experienced faster diffusion on the nanoscale curvature only when embedded in a membrane capable of sustaining lipid phase separation at low temperatures. The curvature-induced sorting and faster diffusion even occurred when the sample temperature was above the miscibility temperature of the planar membrane, implying that the nanoscale curvature could induce phase separation in otherwise homogeneous membranes. Further confirmation and understanding of these results are provided by continuum and coarse-grained molecular dynamics simulations with explicit and spontaneous curvature-phase coupling, respectively. The curvature-induced membrane compositional heterogeneity and altered dynamics were achieved only with a coupling of the curvature with a lipid phase separation. These cross-validating results demonstrate the complex interplay of lipid phases, molecular diffusion, and nanoscale membrane curvature that are critical for membrane functionality.SignificanceNanoscopic membrane organization and dynamics are critical for cellular function but challenging to experimentally measure. This work brings together super-resolution optical methods with multiscale computational approaches to reveal the interplay between curvature, composition, phase, and diffusion in model membranes. We report that curvature can induce phase separation in otherwise homogeneous membranes and that the phase-curvature coupling has a direct implication on lipid mobility. This discovery advances our understanding of the fundamental membrane biophysics that regulate membrane activities such as endocytosis and viral budding.

The cell surface is the playground for rich biological phenomena. These emerge from short-range interactions resulting in the nanoscale organization of membrane proteins and lipids, the conformational dynamics of membrane proteins and their interactions with lipids as well as topological changes in membrane form and structure. Such phenomena cooperate to manifest into important cellular phenomena such as cell adhesion, signal transduction, secretory and endocytic trafficking, and cytoskeletal dynamics. In fact, differences in membrane composition and organization can lead to diseased conditions, even cell death. Lipid membranes are also considered as early signatures of life as they demarcate living matter into cells. Consequently, an understanding of membrane processes requires an appreciation of the membrane from a ‘living matter’ perspective that signals within, into, and out of the cell.

Background and purpose α4β2 nicotinic acetylcholine receptor (nAChR), a subtype of the ligand-gated ion channel, is abundantly expressed in the brain and implicated in several neurological disorders. The endocytosis of nAChR plays important roles in the pathogenesis of neurological diseases, but its molecular mechanisms remain poorly understood. Experimental approach Loss-of-function approaches and mutants of α4β2 nAChR that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signaling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of α4β2 nAChR was determined and crosschecked using an enzyme-linked immunosorbent assay and radioligand assay. Key results Endocytosis of α4β2 nAChR occurred through clathrin-mediated endocytosis in a dynamin-dependent manner. 14-3-3η-dependent Src-mediated phosphorylation of the nAChR α4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of β-arrestin1 and G protein-coupled receptor (GPCR) kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homolog-mediated ubiquitination and subsequent downregulation of α4β2 nAChR. Conclusions and implications α4β2 nAChR, an ionophore receptor, employs the metabotropic signaling pathway required for endocytosis, which leads to ubiquitination and downregulation. Further, GRK2 and β-arrestin1, representative players of GPCRs, are involved in the endocytosis of α4β2 nAChR via different mechanisms. Considering the functional and pathological implications of nAChR endocytosis, results obtained in this study are crucial for the progression of basic research and clinical investigations.

Membrane Type 1 Matrix Metalloprotease (MT1-MMP) contributes to the invasive progression of breast cancers by degrading extracellular matrix tissues. Nucleoside diphosphate kinase, NME1/NM23-H1, has been identified as a metastasis suppressor; however, its contribution to local invasion in breast cancer is not known. Here, we report that NME1 is up-regulated in ductal carcinoma in situ (DCIS) as compared to normal breast epithelial tissues. NME1 levels drop in microinvasive and invasive components of breast tumor cells relative to synchronous DCIS foci. We find a strong anti-correlation between NME1 and plasma membrane MT1-MMP levels in the invasive components of breast tumors, particularly in aggressive histological grade III and triple-negative breast cancers. Knockout of NME1 accelerates the invasive transition of breast tumors in the intraductal xenograft model. At the mechanistic level, we find that MT1-MMP, NME1 and dynamin-2, a GTPase known to require GTP production by NME1 for its membrane fission activity in the endocytic pathway, interact in clathrin-coated vesicles at the plasma membrane. Loss of NME1 function increases MT1-MMP surface levels by inhibiting endocytic clearance. As a consequence, the ECM degradation and invasive potentials of breast cancer cells are enhanced. This study identifies the down-modulation of NME1 as a potent driver of the in situ-to invasive transition during breast cancer progression.

Dent disease 1; Cellular models Enfermedad de Dent 1; Modelos celulares Malaltia de Dent 1; Models cel·lulars Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl−/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype–phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology. This work was supported in part by Asdent Patients Association and grants from Ministerio de Ciencia e Innovación (SAF201459945-R and SAF201789989-R to A.M.), the Fundación Senefro (SEN2019 to A.M.) and Red de Investigación Renal REDinREN (12/0021/0013). A.M. group holds the Quality Mention from the Generalitat de Catalunya (2017 SGR).

Membrane depolarization activates the multisubunit CaV 1.2 L-type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of CaV 1.2-mediated Ca2+ signals. The mechanisms regulating the residency time of the channel at the cell membrane are unclear. Here, we coexpressed the channel core complex CaV 1.2α1 pore-forming and auxiliary CaV β subunits and analyzed their trafficking dynamics from single-particle-tracking trajectories. Speed histograms obtained for each subunit were best fitted to a sum of diffusive and directed motion terms. The same mean speed for the highest-mobility state underlying directed motion was found for all subunits. The frequency of this component increased by covalent linkage of CaV β to CaV 1.2α1 suggesting that high-speed transport occurs in association with CaV β. Selective tracking of CaV 1.2α1 along the postendocytic pathway failed to show the highly mobile state, implying CaV β-independent retrograde transport. Retrograde speeds of CaV 1.2α1 are compatible with myosin VI-mediated backward transport. Moreover, residency time at the cell surface was significantly prolonged when CaV 1.2α1 was covalently linked to CaV β. Thus, CaV β promotes fast transport speed along anterograde trafficking and acts as a molecular switch controlling the endocytic turnover of L-type calcium channels.

Actin filaments play multiple roles in the secretory pathway and endosome dynamics in mammals, including maintenance of Golgi structure, release of membrane cargo from the trans-Golgi network (TGN), endocytosis, and endosomal sorting dynamics. In addition, both TGN carrier transport and endocytosis occur by multiple mechanisms in mammals. Actin likely plays a role in at least four mammalian endocytic pathways (clathrin-mediated, CLIC/GEEC, FEME, and IL2R endocytosis), five pathways for membrane release from the TGN (clathrin-mediated, Rab6-mediated, GOLPH3-mediated, sphingomyelin-enriched, Arf1-mediated), and three processes on endosomes (regulated PM recycling, transport to the trans-Golgi network, and transition from early to late endosomes). Finally, the mammalian Golgi structure is highly dynamic, and actin likely plays roles in these dynamics. One challenge for many of these processes is the need to deal with other membrane-associated structures, such as the cortical actin network on the PM or the matrix that surrounds the Golgi. Arp2/3 complex is a major actin assembly factor in most of the processes discussed, but roles for formins and tandem WH2 motif-containing assembly factors are being elucidated and are anticipated to expand with further study. The specific role for actin is not defined for most of these processes, but is likely to be in generating force for membrane dynamics, either by actin polymerization itself or by myosin motor activity. Defining these processes mechanistically is necessary for understanding membrane dynamics in general, as well as pathways utilizing these processes, such as autophagy.

Extracellular vesicles (EVs) are a heterogeneous group of membrane-limited vesicles and multi-signal messengers loaded with biomolecules. Exosomes and ectosomes are two different types of EVs generated by all cell types. Their formation depends on local microdomains assembled in endocytic membranes for exosomes and in the plasma membrane for ectosomes. Further, EV release is a fundamental process required for intercellular communication in both normal physiology and pathological conditions to transmit/exchange bioactive molecules to recipient cells and the extracellular environment. The unique structure and composition of EVs enable them to serve as natural nanocarriers, and their physicochemical properties and biological functions can be used to develop next-generation nano and precision medicine. Knowledge of the cellular processes that govern EVs biology and membrane trafficking is essential for their clinical applications. However, in this rapidly expanding field, much remains unknown regarding EV origin, biogenesis, cargo sorting, and secretion, as well as EV-based theranostic platform generation. Hence, we present a comprehensive overview of the recent advances in biogenesis, membrane trafficking, and functions of EVs, highlighting the impact of nanoparticles and oxidative stress on EVs biogenesis and release and finally emphasizing the role of EVs as nanotherapeutic agents.

The endosomal-lysosomal system mediates the process of protein degradation through endocytic pathway. This system consists of early endosomes, late endosomes, recycling endosomes and lysosomes. Each component in the endosomal-lysosomal system plays individual crucial role and they work concordantly to ensure protein degradation can be carried out functionally. Dysregulation in the endosomal-lysosomal system can contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). In AD endosomal-lysosomal abnormalities are the earliest pathological features to note and hence it is important to understand the involvement of endosomal-lysosomal dysfunction in the pathogenesis of AD. In-depth understanding of this dysfunction can allow development of new therapeutic intervention to prevent and treat AD.