Pathways and mechanisms of endocytic recycling Grant, Barth D; Donaldson, Julie G
Nature reviews. Molecular cell biology,
200909, 2009-Sep, 2009-09-00, 20090901, Letnik:
10, Številka:
9
Journal Article
Recenzirano
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Endocytic recycling is coordinated with endocytic uptake to control the composition of the plasma membrane. Although much of our understanding of endocytic recycling has come from studies on the ...transferrin receptor, a protein internalized through clathrin-dependent endocytosis, increased interest in clathrin-independent endocytosis has led to the discovery of new endocytic recycling systems. Recent insights into the regulatory mechanisms that control endocytic recycling have focused on recycling through tubular carriers and the return to the cell surface of cargoes that enter cells through clathrin-independent mechanisms. Recent work emphasizes the importance of regulated recycling in processes as diverse as cytokinesis, cell adhesion, morphogenesis, cell fusion, learning and memory.
Macropinocytosis is a form of endocytosis that brings large fluid-filled endosomes into the cell interior. Macrophages and dendritic cells are especially active in this process, but all cells can be ...stimulated to initiate this remarkable form of endocytosis. Although much is known about the membrane lipid and actin requirements for initiating macropinocytosis, less is known about the membrane that forms the macropinosome and the fate of that membrane once the macropinosome enters the cell interior. Since macropinocytosis is a specialized form of clathrin-independent endocytosis (CIE), studies of the constitutive internalization and trafficking of cargo proteins and membrane that enter cells independently of clathrin could reveal the types of membrane that form the macropinosome and the machinery that handles cargo sorting and recycling during the maturation of the macropinosome. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.
Clathrin-independent pathways of endocytosis Mayor, Satyajit; Parton, Robert G; Donaldson, Julie G
Cold Spring Harbor perspectives in biology,
2014-Jun-02, Letnik:
6, Številka:
6
Journal Article
Recenzirano
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There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways ...by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration.
Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide-binding (G) proteins, including the ARF-like (ARL) proteins and SAR1, regulate membrane traffic and organelle structure by ...recruiting cargo-sorting coat proteins, modulating membrane lipid composition, and interacting with regulators of other G proteins. New roles of ARF and ARL proteins are emerging, including novel functions at the Golgi complex and in cilia formation. Their function is under tight spatial control, which is mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that catalyse GTP exchange and hydrolysis, respectively. Important advances are being gained in our understanding of the functional networks that are formed not only by the GEFs and GAPs themselves but also by the inactive forms of the ARF proteins.
Macropinocytosis is an actin-driven form of clathrin-independent endocytosis that generates an enlarged structure, the macropinosome. Although many studies focus on signaling molecules and ...phosphoinositides involved in initiating macropinocytosis, the commitment to forming a macropinosome and the handling of that membrane have not been studied in detail. Here we show in HT1080 cells, a human fibrosarcoma cell line, a requirement for microtubules, dynein, the JIP3 microtubule motor scaffold protein, and Arf6, a JIP3 interacting protein, for the formation and inward movement of the macropinosome. While actin and myosin II also play critical roles in the formation of ruffling membrane, microtubules provide an important tract for initiation, sealing, and transport of the macropinosome through the actin- and myosin-rich lamellar region.
Clathrin independent endocytosis (CIE) is a form of endocytosis present in all cells that mediates the entry of nutrients, macromolecules and membrane proteins into cells. When compared to ...clathrin-dependent endocytosis (CDE), however, much less is known about the machinery involved in forming CIE endosomes. One way to distinguish CIE from CDE has been to deplete cells of coat proteins involved in CDE such as clathrin or the dynamin GTPase, leading to a block of CDE but not CIE. A drawback of such genetic manipulations is that depletion of proteins important for mediating CDE over a period of days can have complex indirect effects on cellular function. The identification of chemical compounds that specifically and rapidly block CDE or CIE would facilitate the determination of whether a process involved CDE or CIE. To date, all of those compounds have targeted CDE. Dynasore and the dynoles specifically target and block dynamin activity thus inhibiting CDE but not most forms of CIE. Recently, a new compound called pitstop 2 was identified as an inhibitor of the interaction of amphiphysin with the amino terminal domain of clathrin, and shown to inhibit CDE in cells. Here we show that pitstop 2 is also a potent inhibitor of CIE. The effects of pitstop 2 are not restricted to inhibition of clathrin since knockdown of clathrin fails to rescue the inhibition of endocytosis of CIE proteins by the drug. Thus pitstop 2 has additional cellular targets besides the amino terminal domain of clathrin and thus cannot be used to distinguish CIE from CDE.
Immunofluorescence Staining Donaldson, Julie G
Current protocols in cell biology,
2015-Dec-01, Letnik:
69
Journal Article
This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules ...for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to a fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescence microscope. This unit describes the widely used and powerful technique of localization of proteins in cells by immunofluorescence. The location can be determined by double labeling with an antibody directed against a protein of known location. The technique can be used as a supplement to immunolocalization by electron microscopy and subcellular fractionation. It allows not only identification of the antigen distribution in the cell but also a survey of the dynamic aspects of protein movements in the cell-on and off membranes, into and out of the nucleus, and through membrane traffic pathways.
Here we show that inhibition of clathrin‐mediated endocytosis does not inhibit clathrin‐independent endocytosis but does alter the endosomal trafficking of cargo proteins that enter cells this way. ...The altered trafficking can be rescued by over‐expression of Rab35 or induced by depletion of Rab35 suggesting that Rab35 activity may serve as an indicator of normal input from clathrin‐mediated endocytosis.
Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.
The discovery that Arf GTPases, mediators of membrane traffic, activate phospholipase D (PLD) raised the possibility that Arfs could facilitate membrane traffic by altering membrane lipid ...composition. PLD hydrolyzes phosphatidylcholine to generate phosphatidic acid (PA), a lipid that favors membranes with negative curvature and thus can facilitate both membrane fission and fusion. This review examines studies that have reported a role for PLD in endocytosis and membrane recycling from endocytic pathways.
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