Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics ...and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.
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•Clathrin-independent endocytosis occurs in response to a decrease in cell volume•Reduced surface tension induces assembly of GRAF1 via the BAR and PH domains•GRAF1 activity suppresses cellular blebbing•Cancer cells lacking GRAF1 are more invasive
Holst et al. show that clathrin-independent endocytosis facilitates the rearrangement of the cell surface in response to a decrease in cell volume. This regulation, mediated by the protein GRAF1, suppresses cellular blebbing and the invasiveness of cancer cells, clarifying why GRAF1 acts as a tumor suppressor.
Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific ...functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-γ and IL-4/IL-13, respectively. M1 and M2 were identified as CD64(+)CD80(+) and CD11b(+)CD209(+), respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-γ, IL-8, TNF-α, IL-1β, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b(+)CD209(+) and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli.
Synaptojanin 1 (SJ1) is a major presynaptic phosphatase that couples synaptic vesicle endocytosis to the dephosphorylation of PI(4,5)P2, a reaction needed for the shedding of endocytic factors from ...their membranes. While the role of SJ1’s 5-phosphatase module in this process is well recognized, the contribution of its Sac phosphatase domain, whose preferred substrate is PI4P, remains unclear. Recently a homozygous mutation in its Sac domain was identified in early-onset parkinsonism patients. We show that mice carrying this mutation developed neurological manifestations similar to those of human patients. Synapses of these mice displayed endocytic defects and a striking accumulation of clathrin-coated intermediates, strongly implicating Sac domain’s activity in endocytic protein dynamics. Mutant brains had elevated auxilin (PARK19) and parkin (PARK2) levels. Moreover, dystrophic axonal terminal changes were selectively observed in dopaminergic axons in the dorsal striatum. These results strengthen evidence for a link between synaptic endocytic dysfunction and Parkinson’s disease.
•Parkinsonism patient mutation in SJ1 produces motor dysfunction in mice•A role of the Sac domain of SJ1 (PARK20) in clathrin coat dynamics at synapses•Selective disruptive impact on a subset of nigrostriatal axons of the SJ1 mutation•Knockin mice for the SJ1 mutation have abnormal levels of PARK2 and PARK19
Cao et al. demonstrate widespread synaptic accumulation of clathrin-coated endocytic intermediates and an additional selective dystrophy of a subset of nigrostriatal dopaminergic axon terminals in knockin mice carrying the synaptojanin 1 (PARK20) mutation responsible for early-onset parkinsonism.
Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here ...we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates.
•CIE mediates the entry of PM proteins with a wide variety of functions.•CIE cargo proteins are sorted on endosomes.•Recruitment of Rab proteins to endosomes regulates CIE cargo trafficking.•CIE is fundamental for cell migration, nutrient metabolism and immune defense.
We previously found that ophthalmic formulations containing nanoparticles prepared by a bead mill method lead to an increase in bioavailability in comparison with traditional formulations (solution ...type). However, the transcorneal penetration pathway for ophthalmic formulations has not been explained yet. In this study, we investigated the mechanism of transcorneal penetration in the application of ophthalmic formulations containing indomethacin nanoparticles (IMC-NPs).
IMC-NPs was prepared by the bead mill method. For the analysis of energy-dependent endocytosis, corneal epithelial (HCE-T) cell monolayers and removed rabbit cornea were thermoregulated at 4°C, where energy-dependent endocytosis is inhibited. In addition, for the analysis of different endocytosis pathways using pharmacological inhibitors, inhibitors of caveolae-mediated endocytosis (54 µM nystatin), clathrin-mediated endocytosis (40 µM dynasore), macropinocytosis (2 µM rottlerin) or phagocytosis (10 µM cytochalasin D) were used.
The ophthalmic formulations containing 35-200 nm sized indomethacin nanoparticles were prepared by treatment with a bead mill, and no aggregation or degradation of indomethacin was observed in IMC-NPs. The transcorneal penetration of indomethacin was significantly decreased by the combination of nystatin, dynasore and rottlerin, and the decreased penetration levels were similar to those at 4°C in HCE-T cell monolayers and rabbit cornea. In the in vivo experiments using rabbits, dynasore and rottlerin tended to decrease the transcorneal penetration of indomethacin (area under the drug concentration - time curve in the aqueous humor AUC
), and the AUC
in the nystatin-treated rabbit was significantly lower than that in non-treatment group. In addition, the AUC
in rabbit corneas undergoing multi-treatment was obviously lower than that in rabbit corneas treated with each individual endocytosis inhibitor.
We found that three energy-dependent endocytosis pathways (clathrin-dependent endocytosis, caveolae-dependent endocytosis and macropinocytosis) are related to the trans-corneal penetration of indomethacin nanoparticles. In particular, the caveolae-dependent endocytosis is strongly involved.
Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in ...the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin‐mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin‐independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N‐BAR domain protein endophilin‐A3. Third, we discovered PSTPIP1, an F‐BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin‐A3 and PSTPIP1, and galectins fine tune the clathrin‐independent endocytosis of L1CAM.
This study reveals that L1CAM is a clathrin‐independent cargo whose endocytosis is controlled by two BAR domain proteins, endophilin‐A3 and PSTPIP1. In addition, galectins regulate L1CAM internalisation.
Phosphorylation of the central adaptor protein complex, AP‐2 is pivotal for clathrin‐mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP‐2 inducible kinase—BMP2K) ...in AP‐2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP‐2 in vitro and in vivo. Functional impairment of BMP2K impedes AP‐2 phosphorylation leading to defects in clathrin‐coated pit (CCP) morphology and cargo internalization. BMP2K engages AP‐2 via its extended C‐terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP‐2 indicating AP‐2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP‐2 loss‐of‐function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP‐2 phosphorylation and in the operation of CME.
AP‐2 is a heterotetrameric adaptor protein complex central to clathrin‐mediated endocytosis (CME). Muniscin family of proteins (muniscins) activates AP‐2 from a closed, inactive state in the cytosol to an open membrane bound state which facilitates clathrin and cargo binding. Here we show that AP‐2 and muniscins likely co‐ordinate the recruitment and stabilization of a previously uncharacterized kinase, BMP2K in clathrin‐coated pits (CCPs). This results in phosphorylation of AP‐2 leading to efficient clathrin‐mediated endocytosis.
Exosomes are extracellular membrane vesicles whose biogenesis by exocytosis of multivesicular endosomes was discovered in 1983. Since their discovery 30 years ago, it has become clear that exosomes ...contribute to many aspects of physiology and disease, including intercellular communication. We discuss the initial experiments that led to the discovery of exosomes and highlight some of the exciting current directions in the field.
Control of membrane traffic: Photoswitchable inhibitors of protein–protein interactions were applied to photoregulate clathrin‐mediated endocytosis (CME) in living cells. Traffic light (TL) peptides ...acting as “stop” and “go” signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation.
Commensal bacteria influence host physiology, without invading host tissues. We show that proteins from segmented filamentous bacteria (SFB) are transferred into intestinal epithelial cells (IECs) ...through adhesion-directed endocytosis that is distinct from the clathrin-dependent endocytosis of invasive pathogens. This process transfers microbial cell wall-associated proteins, including an antigen that stimulates mucosal T helper 17 (T
17) cell differentiation, into the cytosol of IECs in a cell division control protein 42 homolog (CDC42)-dependent manner. Removal of CDC42 activity in vivo led to disruption of endocytosis induced by SFB and decreased epithelial antigen acquisition, with consequent loss of mucosal T
17 cells. Our findings demonstrate direct communication between a resident gut microbe and the host and show that under physiological conditions, IECs acquire antigens from commensal bacteria for generation of T cell responses to the resident microbiota.
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