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Mechanisms and Functions of Endocytosis

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Mechanisms and Functions of Endocytosis JCB: MEETING REVIEW Mechanisms and functions of endocytosis 1 2 Marta Miaczynska and Harald Stenmark 1 Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland 2 Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, and Department of Biochemistry, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway A recent EMBO-FEBS workshop en- CCPs could be distinguished, two short- strong stimulation of neurons. The mor- titled Endocytic Systems: Mechanism lived (early- and late-abortive with life- phology of KO nerve terminals was visu- times in the range of seconds) and one alized by EM tomography followed by and Function, organized by Howard long-lived productive population stable tridimensional reconstruction. Such syn- Riezman in Villars-sur-Ollon (Switzer- for over one minute. Interestingly, cargo apses are fi lled with clusters of clathrin- land), showcased the multifaceted appears to increase a number of produc- coat components, forming tubular networks approaches and model systems used tive, long-lived CCPs/CCVs without af- capped by clathrin-coated pits that open to study endocytosis. The meeting fecting their lifetimes, which can in turn to the plasma membrane. revealed how endocytosis controls be regulated by the activity of dynamin. Morphological heterogeneity of Role of actin in clathrin- multiple aspects of biology, ranging CCVs was emphasized by Tomas Kirch- dependent endocytosis from development to immunity and hausen (Harvard Medical School). Cryo- Because of the ease of genetic manipula- neurotransmission. electron tomography of individual CCVs tions, the yeast Saccharomyces cerevisiae revealed a broad range of patterns used has been very useful for dissecting the to organize a clathrin lattice, with asym- molecular machineries of endocytosis. Mechanisms of clathrin- metrically located membrane vesicles Genetic studies have revealed an essen- dependent endocytosis buried inside the shell ( Cheng et al., 2007 ). tial role for actin in endocytosis in yeast, Although clathrin-mediated internaliza- Moreover, high-resolution imaging of live and a key question concerns how actin tion has been investigated for many years, cells based on total internal refl ection functions together with clathrin in endo- new technologies keep providing us with fl uorescence technology indicates that cytosis. Using real-time image analysis novel insights into its underlying mecha- AP-2 adaptor proteins are also localized of yeast cells expressing fl uorescently nisms, with an unprecedented scale down nonsymmetrically within an individual tagged versions of more than 40 endo- to molecular details regarding the struc- CCV. This may result from an initially cytic proteins, David Drubin (University ture and dynamics of the proteins involved. restricted localization of adaptors, as of California, Berkeley) has analyzed the The plasticity of clathrin-mediated inter- they are captured during the nucleation dynamic appearance, movement, and nalization was well illustrated by the fact and early phases of coated pit assembly, disappearance of these proteins at endo- that this pathway is used not only to traffi c while retaining the adaptors concen- cytic sites. Drubin presented data indi- extracellular molecules or cellular pro- trated at the place of their original recruit- cating that these proteins can be grouped ment at the time of vesicle pinching and into four functional modules that medi- THE JOURNAL OF CELL BIOLOGY teins, but also to mediate entry of certain toxins, viruses, and bacteria. CCV formation. ate coat formation, membrane invagina- The issue of lifetimes of clathrin- Clathrin-mediated endocytosis tion, actin-meshwork assembly, and vesicle coated pits (CCPs) and vesicles (CCVs) serves some specialized functions in var- scission during clathrin/actin-mediated has remained controversial, as values ious tissues, including the nervous sys- endocytosis. Maria-Isabel Geli (Instituto reported in the literature range from sec- tem. Knockout (KO) studies in mice, de Biolog í a Molecular de Barcelona) de- onds to minutes. Sandra Schmid (The reported by Pietro De Camilli (Yale scribed an in vitro assay to reconstitute Scripps Research Institute) described University School of Medicine), demon- the complex actin structures that partici- quantitative computational analyses to strated that dynamin-1 appeared not pate in the formation of endocytic profi les track the dynamics of CCP/CCV forma- to be essential for the biogenesis and and the use of immuno-electron micros- tion on the plasma membrane. In this way, endocytic recycling of synaptic vesicles copy to define the primary endocytic three kinetically distinct populations of ( Ferguson et al., 2007 ), although studies profi les in yeast and the localization of of dynamin mutants in cultured cells the actin machinery. would have predicted a crucial role for Given the importance of actin and this protein in vivo. The role of dynamin-1 clathrin in endocytosis, proteins that link Correspondence to Marta Miaczynska: miaczynska @iimcb.gov.pl; or Harald Stenmark: stenmark@ in synaptic vesicle endocytosis is activity actin and clathrin functions are of special ulrik.uio.no dependent and becomes evident during interest. Genetic studies in yeast have © The Rockefeller University Press $30.00 The Journal of Cell Biology, Vol. 180, No. 1, January 14, 2008 7–11 http://www.jcb.org/cgi/doi/10.1083/jcb.200711073 JCB 7 indicated that clathrin light chain may regulate the ability of Sla2 to control ac- tin dynamics in endocytosis ( Newpher et al., 2006 ). Frances Brodsky (Univer- sity of California, San Francisco) de- scribed a study of Hip1 and Hip1R, the mammalian homologues of Sla2, which have overlapping but not identical func- tions in endocytosis. Brodsky presented evidence that Hip proteins interact se- quentially with clathrin and actin rather than functioning as bridges between the two. Clathrin-dependent endocytosis and pathogen entry Certain toxins and pathogens harness clathrin-mediated internalization to enter cells. Endocytosis of anthrax toxin, de- scribed by Gisou van der Goot (Federal Polytechnic School of Lausanne), is Figure 1. Distinct endocytic internalization pathways discussed at the conference, with examples of clathrin- and dynamin-mediated but key regulators. Color code: blue, coat proteins; red, kinases; orange, GTPases; green, adaptors; requires also the presence of lipid rafts, a black, other proteins. The various functions of endocytosis are indicated on top. classical hallmark of clathrin-independent entry routes. The protective antigen (PA) However, the clathrin-mediated involved and cargos entering via these subunit of the toxin binds to cell surface pathway appears to be exploited not only mechanisms. This allows us now to re- receptors (TEM8 and CMG2) and induces by viruses, but also by much larger bac- defi ne these pathways more precisely and their multiple post-translational modifi - terial pathogens such as Listeria mono- in positive terms, in contrast to their initial cations, such as palmitoylation, phos- cytogenes, as reported by Pascale Cossart collective description as “ non-clathrin phorylation, and ubiquitination, which (Pasteur Institute of Paris). In a process endocytosis ” . Some classifi cation schemes differentially regulate toxin internaliza- induced by the bacterial surface protein have been already proposed ( Mayor and tion ( Abrami et al., 2006 ). Yet another InlB, clathrin and auxilin are recruited Pagano, 2007 ), based primarily on the new player implicated in this process around the entering bacteria, followed dependence on dynamin and various small appears to be the Wnt coreceptor LRP6, by actin polymerization. Nevertheless, GTPases; however, the exact number of which interacts with TEM8 and CMG2 despite certain similarities and common clathrin-independent pathways and their and its depletion results in reduced toxin players involved (dynamin, Eps15, and mutual relations remain unclear. uptake. Anthrax toxin thus provides an the E3 ubiquitin ligase Cbl), this process Satyajit Mayor (National Centre for interesting example of using complex appears mechanistically and kinetically Biological Sciences, Bangalore) reported intracellular endocytic and signaling different from the canonical clathrin- progress toward further characterization mechanisms for precise regulation of its mediated internalization of macro- of a clathrin-, dynamin-, and caveolae- internalization in time and space. molecules. This in turn argues that the independent internalization route that Semliki forest virus (SFV) was one networks of protein – protein and protein– transports GPI-anchored proteins (GPI- of the fi rst viral pathogens identifi ed to lipid interactions involved in clathrin- APs) and involves GEECs (GPI-AP – exploit clathrin-dependent internalization mediated internalization can assemble in enriched early endosomal compartments) mode, as recalled by Ari Helenius (ETH various combinations. This plasticity may as intermediates. This constitutive pino- Zurich) in his plenary lecture, along with be exploited not only by pathogens, but cytic pathway is initiated by a cholesterol- a number of viruses using other pathways also under physiological conditions by dependent recruitment and stabilization (see Fig. 1 ). Further viruses, such as spe- different types of cells or tissues with of active Cdc42 on the plasma mem- cies C adenovirus type 2 (Ad2) and Ad5 particular needs for specialized forms brane,
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