Based on protein–protein interactions, the synaptic dynamin-associated protein, SdpI, has been proposed to link actin dynamics and endocytic processes in the nerve terminal (Qualmann et al. 1999
). Whereas SdpI is brain-specific, SdpII, described here, exhibits a wide tissue distribution. The colocalization of endogenous SdpII with dynamin in PC12 cells is consistent with a role in endocytosis. The behavior of cells transfected with either of the two syndapins or their fragments, the distribution of exogenous syndapin, and the interaction of SdpI and -II with synaptojanin, dynamin I, synapsin I, and N-WASP strongly suggest a center role for the syndapins in linking endocytosis to the actin cytoskeleton. The broad tissue distribution of SdpII and the existence of syndapin-related proteins in a wide array of multicellular organisms (Echinococcus
, Caenorhabditis elegans
, rat, mouse, and human) furthermore suggest that a connection of endocytosis and actin organization via syndapin is based on a conserved machinery in many different organisms and cell types.
To establish a function of syndapin in endocytosis, we tested the physiological significance of the determined syndapin protein interactions by assaying the effect of overexpression of both SdpI and -II SH3 domains on transferrin uptake. We observed a potent block in receptor-mediated endocytosis in vivo. This is in striking contrast to the SH3 domains of Grb2, spectrin, and phospholipase Cγ that bind to dynamin in vitro, but do not block receptor-mediated endocytosis (Wang and Moran 1996
; Wigge et al. 1997
). Therefore, both syndapin isoforms appear to play a role in clathrin-mediated endocytosis, although SdpI, which is expressed at much higher levels in brain, might be specialized for synaptic vesicle endocytosis. The transfection studies are consistent with earlier in vitro reconstitution assays in which the SdpI SH3 domain interfered with endocytic coated vesicle formation (Simpson et al. 1999
). The reconstitution studies also suggested that the interaction of the syndapin SH3 domain with one of its partners is required for late events in endocytosis, leading to vesicle fission. Late events in coated vesicle formation were also selectively affected by a dynamin mutant defective in GTP binding and the actin monomer sequestering drugs, latrunculin B and thymosin beta4 (Damke et al. 1994
; Lamaze et al. 1997
Syndapins interact with synaptojanin, synapsin I, and N-WASP, all proteins implicated in cytoskeletal reorganization (Bähler and Greengard 1987
; Miki et al. 1996
; Sakisaka et al. 1997
). In this study, we have provided in vivo evidence that syndapin modulates the actin cytoskeleton. Syndapin overexpression triggered the formation of filopodia, protrusions from the cell surface containing bundled actin filaments that are found particularly in motile cells and at the ends of growth cones in neurons (O'Connor and Bentley 1993
). Full-length syndapin proteins, but not the SH3 domain or the NH2
terminus alone, induced filopodia upon overexpression. Strikingly, syndapin localizes to the very tips of filopodia induced upon overexpression, which represent the actual sites of actin polymerization (Okabe and Hirokawa 1991
) and to the edges of lamellipodia. Neither location is a known site of endocytosis.
Our data suggest that syndapin-induced cortical actin rearrangements are mediated by the Arp2/3 complex. First, syndapin and Arp3 colocalize at lamellipodial structures induced upon syndapin overexpression. Likewise, GFP–dynamin 2, but not other parts of the endocytosis machinery, like clathrin, has been reported to localize to cortical membrane ruffles and lamellipodia (Cao et al. 1998
). Second, coexpression of a COOH-terminal fragment of N-WASP completely suppressed syndapin-triggered filopodia formation (). An analogous fragment of Scar or WASP was shown by Machesky and Insall 1998
to impair cortical localization of the Arp2/3 complex and lamellipodia formation. Thus, syndapin-induced filopodia formation appears to require the Arp2/3 complex and its translocation to the cell cortex. In contrast, filopodia formation triggered by constitutively active Cdc42 mutants was not inhibited by the coexpression of the Scar COOH terminus (Machesky and Insall 1998
). This is consistent with the observation that filopodia induced by membrane recruitment of Cdc42 differed from those induced by WASP in morphology and protein composition (Castellano et al. 1999
), suggesting that there may be two types of filopodia formation and syndapins may only induce one class.
Overexpression of the dynamin K44A mutant (Damke et al. 1994
) and K+
-depletion or cytosolic acidification (Altankov and Grinnell 1993
) were shown to block receptor-mediated endocytosis, but also to alter cell shape and redistribute actin stress fibers. In these cases, the defects in cell morphology and cytoskeletal organization might be secondary to an endocytosis block. We have shown here that different domains of syndapin affect membrane trafficking and cytoskeletal architecture in vivo. While the SH3 domain alone was sufficient to block endocytosis, it did not induce filopodia. To trigger these actin rearrangements, overexpression of the full-length protein was necessary, showing that the syndapin-induced cytoskeletal rearrangements, at least, are not an indirect consequence of the inhibition of receptor-mediated endocytosis.
Several potential mechanisms may explain the syndapin-induced rearrangement of the cortical actin cytoskeleton. First, syndapin may mimic N-WASP activators, such as Cdc42 (Rohatgi et al. 1999
), causing a conformational change of N-WASP, which allows its COOH terminus to interact with and stimulate the Arp2/3 complex actin polymerization machinery. Second, syndapin may recruit N-WASP to the plasma membrane and this recruitment by itself may be sufficient for filopodia formation. In support of this, overexpression of N-WASP alone was insufficient to alter cell morphology or cytoskeletal architecture (Miki et al. 1998
), whereas the local recruitment of WASP to a membrane receptor resulted in the formation of membrane protrusions (Castellano et al. 1999
). A third potential mechanism is syndapin could act upstream of Cdc42 and activate the GTPase. And fourth, syndapin itself may have actin-modulating properties in conjunction with the Arp2/3 complex.
In general, the actin cytoskeleton could be involved in endocytic processes in several different ways, implicating different potential functional roles for syndapin. Cytoskeletal structures may help to organize the endocytic machinery at the plasma membrane. Specific sites, or “hot spots”, for synaptic vesicle recycling have been observed in Drosophila
nerve terminals (Estes et al. 1996
; González-Gaitán and Jäckle 1997
; Roos and Kelly 1998
). Since syndapin protein family members represent multidomain proteins, they could provide the linkage between the endocytic machinery and the cytoskeleton. A rigid cortical actin cytoskeleton has an inhibitory effect on membrane traffic (Trifaró and Vitale 1993
). Syndapin might thus, via its interaction with N-WASP, promote local actin treadmilling, removing physical barriers to endocytosis. A third potential function of the dynamin-associated syndapin involves the membrane fission event. By in vitro reconstitution studies, syndapin was implicated in the late, coated-pit scission event (Simpson et al. 1999
), which is also selectively affected by a dynamin mutant (Damke et al. 1994
) and by actin monomer sequestering drugs in mammalian A431 cells (Lamaze et al. 1997
). Perhaps the actin cytoskeleton provides the force to drive membrane fission. Actin polymerization may also promote the movement of newly formed endocytic vesicles into the cytoplasm. Both pinosomes and clathrin-coated vesicles have recently been described associated with actin comet tails in the cytoplasm (Frischknecht et al. 1999
; Merrifield et al. 1999
). Finally, it is possible that syndapin regulation allows either endocytosis to take place at a patch of membrane or actin rearrangement, but not both simultaneously.
Our results strongly suggest that SdpI links endocytosis and cytoskeletal dynamics in mature nerve terminals, whereas SdpII performs a similar role in other cell types.