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 The Syndapin protein family ? differentially controlled proteins at the interface of endocytosis, actin cytoskeleton and signaling

( Regina Dahlhaus, Rashmi Ahuja, Anne Braun, Dennis Koch)

The biochemical and functional characterization of syndapin proteins strongly suggests them to have both endocytic and cytoskeletal functions, i.e. to represent functional links between membrane trafficking and the cortical actin cytoskeleton (Figure Project 1.1; Qualmann et al., 1999; Qualmann & Kelly, 2000).

Overexpression of syndapin induces actin-rich filopodia.

Fig. 1: Overexpression of syndapin induces actin-rich filopodia. Syndapin (green) induces actin-rich filopodia (stained with phalloidin; red) upon overexpression in HeLa cells. Untransfected cell show no filopodia at the cell surface.

We have hypothesized that and by which mechanisms the cortical actin cytoskeleton may support endocytic vesicle formation (Qualmann et al., 2000). These ideas are strongly supported by the recent observation of local actin polymerization at sites of endocytosis and its correlation with vesicle fission mediated by the GTPase dynamin in time and space (Merrifield et al., 2002, Nat. Cell Biol. 4, 691-8). In an attempt to unravel the molecular mechanism involved, we have focussed our recent studies on the interaction of syndapins with the neuronal Wiskott-Aldrich-Syndrom protein (N-WASP), a potent stimulator of a prominent cellular actin polymerization machinery (the Arp2/3 complex). We have interfered specifically with the N-WASP/syndapin interaction or with N-WASP functions in general in living cells and observed in all cases a potent inhibition of receptor-mediated endocytosis. Importantly, we were able to restore endocytic function by corresponding rescue experiments. Our studies revealed that both syndapins and N-WASP are crucial components of the vesicle formation machinery (Kessels & Qualmann, 2002).

Since syndapins interact with dynamin, i.e. the molecular player for vesicle fission control, it seemed possible that syndapins interconnect fission control with actin polymerization mediated by the Arp2/3 complex activator N-WASP at sites of endocytosis (Figure 2).

Syndapins link endocytic vesicle formation and the actin cytoskeleton.

Fig. 2: Syndapins link endocytic vesicle formation and the actin cytoskeleton. Syndapins interact with both the GTPase dynamin that mediates vesicle formation processes at the plasma membrane and with the neuronal Wiskott-Aldrich-Syndrom protein (N-WASP), a potent stimulator of the prominent cellular actin polymerization machinery, as shown in vitro and in vivo. Interfering with dynamin functions by overexpression of the dynamin-binding module of syndapins (such cells appear in red) blocks receptor-mediated endocytosis that can be followed by a green fluorescent tracer (left microscopic image). Overexpression of syndapins as full-length proteins (in the cells marked by stars in the right microscopic image) elicits a striking reorganization of the cortical cytoskeleton, the induction of numerous actin-rich, thin protrusions from the surface of the cells. Adapted from Qualmann & Kelly, 2000, J. Cell Biol. 148, 1047-62.

In order to fulfill such a role, however, syndapins would have to be able to recruit N-WASP to the membrane. By permanently attaching syndapins to intracellular membranes we were indeed able to reconstitute and observe this property in vivo . Since these experiments also led to local actin polymerization, membrane-bound protein complexes composed of syndapins, N-WASP and the Arp2/3 complex seem be able to trigger local actin polymerization (Kessels & Qualmann, 2002). Such a local actin polymerization may be a mechanism that supports vesicle detachment and movement away from the plasma membrane. Our most recent data in collaboration with Prof. Almers and Dr. Merrifield using high resolution evanescent filed microscopy reveal that transient actin polymerization at clathrin-coated structures leaving the membrane coincides with a local recruitment of the Arp2/3 complex actin polymerization machinery (Merrifield et al, 2004). Our high resolution life imaging analyses furthermore revealed that internalization of clathrin-coated pits is not only accompanied by a recruitment of the Arp2/3 complex but also by the Arp2/3 complex activator N-WASP and that the main recruitment of N-WASP was observed right upon clathrin departure (Merrifield et al., 2004). These observations support our hypothesis that local actin polymerization involving the Arp2/3 complex and its activator N-WASP may be a mechanism supporting clathrin-coated vesicle detachment and movement away from the plasma membrane (Kessels & Qualmann, 2004).

The observed coupling between actin and membrane trafficking might be of particular importance in cells that rely on very efficient and highly regulated membrane trafficking processes, such as regulated secretory cells. In collaboration with the Sarah Hamm-Alvarez lab we therefore studied membrane trafficking and the cytoskeleton at the apical side of lacrimal acini cells (da Costa et al., 2003). These cells are the principal source of tear proteins released into the nascent tear fluid. Much alike neurons, these cells perform massive exocytosis and the required compensatory endocytosis. We observed that promoting aberrant actin polymerization blocked endocytosis. Also the opposite manipulation, block of actin polymerization and promotion of depolymerization, led to an endocytosis inhibition. We can thus conclude that, in these specialized secretory cells, apical endocytosis relies on undisturbed actin dynamics - a cellular function that includes both polymerization and depolymerization (da Costa et al., 2003).

Our most recent examinations reveal that vesicle formation at the plasma membrane, from the recycling endosome and from the Golgi may mechanistically actually be much more similar than previously thought. Similar to the plasma membrane, the Golgi is marked by membrane-associated actin cytoskeletal structures that do not only appear crucial for Golgi positioning and morphology but seem also to participate in membrane transport processes originating from the Golgi (Kessels & Qualmann, 2005). Exit from the recycling compartment also requires transport vesicle generation. Relatively little is known about the molecular mechanisms by which vesicles are formed from recycling endosomes. Among the few components identified to be crucial for recycling are EHD proteins, proteins that bind nucleotides and contain a C-terminal eps15 homology (EH) domain. Interestingly, syndapins interact with EHD proteins and our detailed examinations showed that syndapin/EHD protein complexes play a crucial role in receptor recycling and revealed how such complexes are modulated (Braun et al., 2005). Since the EHD protein interactions are SH3 domain independent, it seems possible that syndapins interconnect EHD protein functions in recycling with functions of components of the actin cytoskeleton and/or the dynamin-containing vesicle fission machinery. The formation of multimeric syndapin complexes and their differential interactions and regulations highlights a molecular mechanism for the hypothesized interconnection and coordination of several molecular machineries working together in vesicle formation processes from different donor membranes within the cell.
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