Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132:617–634). Based on these results, dynactin was proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes (labeled with antitransferrin receptor), as well as late endosomes and lysosomes (labeled with anti–lysosome-associated membrane protein-1 [LAMP-1]), were redistributed to the cell periphery. This redistribution was disrupted by nocodazole, implicating an underlying plus end–directed microtubule motor activity. The Golgi stack, monitored using sialyltransferase, galactosyltransferase, and N-acetylglucosaminyltransferase I, was dramatically disrupted into scattered structures that colocalized with components of the intermediate compartment (ERGIC-53 and ERD-2). The disrupted Golgi elements were revealed by EM to represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A was not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment revealed that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor–cargo binding.

Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.CD95 (APO-1/Fas) is the best-studied member of the death receptor family (26). We previously demonstrated that CD95 oligomerizes upon triggering, forming sodium dodecyl sulfate (SDS)-stable microaggregates on SDS-polyacrylamide gel electrophoresis (PAGE) (11). This activated receptor recruits the adapter molecule FADD and the initiator caspase 8 to form the death-inducing signaling complex (DISC) (11). Recently, Siegel et al. (37) refined this model by showing that unstimulated CD95 exists as preassociated complexes, and they and others (10, 24) confirmed the initial observation of the formation of SDS-stable aggregates by stimulated CD95 (11). In addition, CD95 has been reported to form clusters at the cell surface in a ligand-dependent fashion either late (43) or, in two other reports, very early (6, 9) after receptor triggering. The relationship between or the kinetic order of all these eventspreassociation, formation of SDS-stable microaggregates, formation of the DISC, and the appearance of higher-order receptor clusters, as seen by immunofluorescence microscopy-is unknown.We have previously described two different CD95 apoptosis pathways (32). In type I cells, caspase 8 is recruited to the DISC, resulting in release of active caspase 8 in quantities sufficient to directly activate caspase 3 (40). However, in type II cells, despite similar expression levels of surface CD95 and signaling molecules, formation of the DISC is so inefficient that only very small quantities of caspase 8 are generated at the cell surface. This amount of caspase 8 is insufficient to process caspase 3, but sufficient to cleave the BH3-only protein Bid (13,16,19), resulting in the apoptogenic activation of mitochondria. Therefore, the execution of apoptosis can be inhibited by overexpression of Bcl-2 or Bcl-x L only in type II cells (32). Recently, a number of transgenic and knockout studies have provided evidence for the existence of the two pathways in vivo (14,17,30,41,48,49). In all cases, CD95 apoptosis execution of thymocytes and peripheral T cells was independent of mitochondria, identi...

T cell cytoarchitecture differs dramatically depending on whether the cell is circulating within the bloodstream, migrating through tissues, or interacting with antigen-presenting cells. The transition between these states requires important signaling-dependent changes in actin cytoskeletal dynamics. Recently, analysis of actin-regulatory proteins associated with T cell activation has provided new insights into how T cells control actin dynamics in response to external stimuli and how actin facilitates downstream signaling events and effector functions. Among the actin-regulatory proteins that have been identified are nucleation-promoting factors such as WASp, WAVE2, and HS1; severing proteins such as cofilin; motor proteins such as myosin II; and linker proteins such as ezrin and moesin. We review the current literature on how signaling pathways leading from diverse cell surface receptors regulate the coordinated activity of these and other actin-regulatory proteins and how these proteins control T cell function.

Actomyosin dynamics and T cell receptor signaling are tightly coupled to ensure proper dynamics and function of signaling microclusters within the immunological synapse.