SummaryFlotillins are membrane proteins that form microdomains in the plasma membrane of all mammalian cell types studied to date. They span the evolutionary spectrum, with proteins related to flotillins present in bacteria, fungi, plants and metazoans, which suggests that they perform important, and probably conserved, functions. Flotillins have been implicated in myriad processes that include endocytosis, signal transduction and regulation of the cortical cytoskeleton, yet the molecular mechanisms that underlie flotillin function in these different cases are still poorly understood. In this Commentary, we will provide an introduction to these intriguing proteins, summarise their proposed functions and discuss in greater detail some recent insights into the role of flotillin microdomains in endocytosis that have been provided by several independent studies. Finally, we will focus on the questions that are raised by these new experiments and their implications for future studies.

Macroautophagy is a mechanism employed by eukaryotic cells to recycle non-essential cellular components during starvation, differentiation, and development. Two conjugation reactions related to ubiquitination are essential for autophagy: Apg12p conjugation to Apg5p, and Apg8p conjugation to the lipid phosphatidylethanolamine. These reactions require the action of the E1-like enzyme, Apg7p, and the E2-like enzymes, Apg3p and Apg10p. In Dictyostelium, development is induced by starvation, conditions under which autophagy is required for survival in yeast and plants. We have identified Dictyostelium homologues of 10 budding yeast autophagy genes. We have generated mutations in apg5 and apg7 that produce defects typically associated with an abrogation of autophagy. Mutants are not grossly affected in growth, but survival during nitrogen starvation is severely reduced. Starved mutant cells show little turnover of cellular constituents by electron microscopy, whereas wild-type cells show significant cytoplasmic degradation and reduced organelle number. Bulk protein degradation during starvation-induced development is reduced in the autophagy mutants. Development is aberrant; the autophagy mutants do not aggregate in plaques on bacterial lawns, but they do proceed further in development on nitrocellulose filters, forming defective fruiting bodies. The autophagy mutations are cell autonomous, because wild-type cells in a chimaera do not rescue development of the autophagy mutants. We have complemented the mutant phenotypes by expression of the cognate gene fused to green fluorescent protein. A green fluorescent protein fusion of the autophagosome marker Apg8 mislocalizes in the two autophagy mutants. We show that the Apg5-Apg12 conjugation system is conserved in Dictyostelium.Protein turnover in eukaryotes is accomplished by two major mechanisms, autophagy or proteasomal degradation. Three modes of autophagy have been identified: chaperone-mediated autophagy, microautophagy, and macroautophagy. In chaperone-mediated autophagy, specific proteins containing targeting sequences are bound by chaperones that mediate direct transport across the lysosomal membrane (1). A lysosomal receptor, lysosomal-associated membrane protein type 2a, interacts with

Macroautophagy is the major mechanism that eukaryotes use to recycle cellular components during stressful conditions. We have shown previously that the Atg12-Atg5 conjugation system, required for autophagosome formation in yeast, is necessary for Dictyostelium development. A second conjugation reaction, Aut7/Atg8 lipidation with phosphatidylethanolamine, as well as a protein kinase complex and a phosphatidylinositol 3-kinase complex are also required for macroautophagy in yeast. In this study, we characterize mutations in the putative Dictyostelium discoideum orthologues of budding yeast genes that are involved in one of each of these functions, ATG1, ATG6, and ATG8. All three genes are required for macroautophagy in Dictyostelium. Mutant amoebae display reduced survival during nitrogen starvation and reduced protein degradation during development. Mutations in the three genes produce aberrant development with defects of varying severity. As with other Dictyostelium macroautophagy mutants, development of atg1-1, atg6؊ , and atg8 ؊ is more aberrant in plaques on bacterial lawns than on nitrocellulose filters. The most severe defect is observed in the atg1-1 mutant, which does not aggregate on bacterial lawns and arrests as loose mounds on nitrocellulose filters. The atg6 ؊ and atg8 ؊ mutants display almost normal development on nitrocellulose filters, producing multi-tipped aggregates that mature into small fruiting bodies. The distribution of a green fluorescent protein fusion of the autophagosome marker, Atg8, is aberrant in both atg1-1 and atg6 ؊ mutants.In the social amoeba Dictyostelium discoideum, starvation is a signal for the initiation of multicellular development. Starving amoebae aggregate in response to cAMP to form mounds. Within these cell aggregates, intercellular signals direct the formation of a multicellular slug that migrates to a suitable location for formation of a fruiting body. The fruiting body is composed of a spore mass held aloft on a stalk composed of cells that vacuolate and die. Development is an energy-intensive process and requires that amoebae cease production of growthrelated proteins and lipids and initiate a developmental program (reviewed in Ref. 1). One mechanism employed by Dictyostelium and other eukaryotes to mobilize resources required for development is macroautophagy. Macroautophagy is required for sporulation in Saccharomyces cerevisiae (2), differentiation in the yeast Podospora anserina (3), metamorphosis in Drosophila melanogaster (4), and dauer development in Caenorhabditis elegans (5). In this transport process, bulk cytoplasm and organelles are sequestered in double-membrane vesicles (autophagosomes/autophagic vacuoles) that fuse with and deliver their content to the lytic compartment of the cell, the lysosome or vacuole. Genetic studies in S. cerevisiae have identified 15 APG genes that are required for the formation of these double membrane autophagosomes (2, 6, 7). A new unified nomenclature for autophagy-related genes was introduced recently (8), and we will ...

The association between flotillin microdomains and the cortical cytoskeleton controls myosin IIa activation and neutrophil chemotaxis.

SummaryThe Gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of freeliving amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum , and macroautophagy genes have been identified and mutated in D. discoideum , we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila . Mutation of the apg1 , apg5 , apg6 , apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D. discoideum . Furthermore, a green fluorescent protein (GFP)-tagged marker of autophagosomes, Apg8, does not systematically colocalize with DsRed-labelled L. pneumophila . We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum .

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In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

When starved, the amoebae of Dictyostelium discoideum initiate a developmental process that results in the formation of fruiting bodies in which stalks support balls of spores. The nutrients and energy necessary for development are provided by autophagy. Atg1 is a protein kinase that regulates the induction of autophagy in the budding yeast Saccharomyces cerevisiae. In addition to a conserved kinase domain, Dictyostelium Atg1 has a C-terminal region that has significant homology to the Caenorhabditis elegans and mammalian Atg1 homologues but not to the budding yeast Atg1. We investigated the function of the kinase and conserved C-terminal domains of D. discoideum Atg1 (DdAtg1) and showed that these domains are essential for autophagy and development. Kinase-negative DdAtg1 acts in a dominant-negative fashion, resulting in a mutant phenotype when expressed in the wild-type cells. Green fluorescent protein-tagged kinase-negative DdAtg1 colocalizes with red fluorescent protein (RFP)-tagged DdAtg8, a marker of preautophagosomal structures and autophagosomes. The conserved C-terminal region is essential for localization of kinase-negative DdAtg1 to autophagosomes labeled with RFP-tagged Dictyostelium Atg8. The dominant-negative effect of the kinase-defective mutant also depends on the C-terminal domain. In cells expressing dominant-negative DdAtg1, autophagosomes are formed and accumulate but seem not to be functional. By using a temperature-sensitive DdAtg1, we showed that DdAtg1 is required throughout development; development halts when the cells are shifted to the restrictive temperature, but resumes when cells are returned to the permissive temperature.Dictyostelium discoideum amoebae have an elaborate program of development that begins with the aggregation of many thousands of amoebae by chemotaxis, followed by the formation of a mound of adhering cells. The mound transforms into a motile slug, which forms a fruiting body consisting of a spore mass on a thin stalk (16). Development occurs only during starvation, and therefore there must be turnover of macromolecules to provide energy and chemical constituents to the developing cells (40).In the face of starvation most eukaryotic cells induce a process of self-digestion called macroautophagy (hereafter, autophagy). Dictyostelium amoebae starving in a nitrogen-free medium survive with near 100% viability for 2 weeks (26,28). Their survival in the nitrogen-free medium depends on the process of autophagy. The events of autophagy begin with a preautophagosomal structure that forms a double membrane autophagosome, which envelops cytoplasm and organelles. These autophagosomes become digestive vacuoles by fusion with a hydrolase-containing vacuole or lysosomes.By mutating the genes that code for Dictyostelium homologues of the budding yeast ATG1, ATG5, ATG6, ATG7, and ATG8, we have shown that autophagy is essential for development and is probably coordinated with it (26, 28). A previously identified Dictyostelium gene called tipD is the homologue of Atg16L, the mammalia...