SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.

Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6⌬ cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition. INTRODUCTIONFormins are key regulators of the actin cytoskeleton and form a large family conserved in all eukaryotes Faix and Grosse, 2006). These proteins are necessary for the formation of numerous actin structures, including stress fibers, filopodia, cytokinetic actin rings, junctional actin structures, or actin cables. The proper regulation of formins is likely to be critical for cellular processes such as cell migration, cytokinesis, cell adhesion, and cell polarity.A well characterized biochemical activity of formins is to nucleate and elongate linear actin filaments Sagot et al., 2002b;Kovar et al., 2003;Li and Higgs, 2003;Moseley et al., 2004). This activity occurs through the formin-homology (FH) 2 domain, which dimerizes to form a doughnut-shaped structure containing multiple actin-binding sites in its core Otomo et al., 2005b). This dimer is thought to stabilize otherwise unstable intermediates in the assembly of new actin filaments, and it binds processively to the fast-elongating barbed end of existing actin filaments (Pring et al., 2003;Kovar and Pollard, 2004).The adjacent FH1 domain binds profilin-actin and helps accelerate the elongation of actin filaments (Chang et al., 1997;Evangelista et al., 1997;Watanabe et al., 1997;Romero et al., 2004;Kovar et al., 2006). The budding yeast formin Bni1p also binds the actin monomer-binding protein Bud6p, which, similar to profilin, stimulates the activity of the FH2 domain (Moseley and Goode, 2005). In addition to their activity in actin filament nucleation and elongation, some formins have been suggested to also function in actin bundling and severing, further contributing to the remodeling of actin structures (Harris et al., 2004Moseley and Goode, 2005;Harris et al., 2006...

The interleukin (IL)‐3 family of cytokines mediates its numerous effects on myeloid growth and maturation by binding a family of related receptors. It has been shown recently that IL‐3 induces the activation of two distinct cytoplasmic signal transducing factors (STFs) that are likely to mediate the induction of immediate early genes. In immature myeloid cells, IL‐3 activates STF‐IL‐3a, which comprises two tyrosine‐phosphorylated DNA binding proteins of 77 and 80 kDa. In mature myeloid cells, IL‐3 and granulocyte‐macrophage colony‐stimulating factor activate STF‐IL‐3b, which consists of a 94 and 96 kDa tyrosine‐phosphorylated DNA binding protein. Peptide sequence data obtained from the purified 77 and 80 kDa proteins (p77 and p80) indicate that they are closely related but are encoded by distinct genes. Both peptide and nucleotide sequence data demonstrate that these two proteins are the murine homologs of ovine mammary gland factor (MGF)/Stat5. The peptide data also indicate that p77 and p80 are phosphorylated on tyrosine 699, a position analogous to the tyrosine that is phosphorylated in Stat1 and Stat2 in response to interferon. Additionally, antiserum raised against bacterially expressed p77/p80 recognizes the 94 and 96 kDa protein components of STF‐IL‐3b, suggesting that these may be additional isoforms of Stat5. These studies indicate that the IL‐3 family of ligands is able to activate multiple isoforms of the signal transducing protein Stat5.

During endocytosis, actin-dependent forces are needed to oppose internal turgor pressure for invagination of the plasma membrane. Live-cell imaging shows that addition of sorbitol to the medium significantly accelerates early steps in the endocytic process and rescues defects of endocytic mutants in fission yeast.

Background Microtubules (MTs) participate in the spatial regulation of actin-based processes such as cytokinesis and cell polarization [1]. The fission yeast Schizosaccharomyces pombe is a rod-shaped cell which exhibits polarized cell growth at cell tips. MT plus ends contact and shrink from the cell tips and contribute to polarity regulation. Results Here, we investigate the effects of changing cell shape on MTs and cell polarization machinery. We physically bend fission yeast cells by forcing them into microfabricated femtoliter chambers. In these bent cells, MTs maintain a straight axis and contact and shrink from cortical sites at the sides of cells. At these ectopic sites, polarity factors such as bud6p, for3p (formin), and cdc42p are recruited and assemble actin cables in an MT-dependent manner. MT contact at the cortex induces the appearance of a bud6p dot within seconds. The accumulation of polarity factors leads to cell growth at these sites, when the MT-associated polarity factor tea1p is absent. This process is dependent on MTs, mal3p (EB1), moe1p (an EB1-binding protein), and for3p, but surprisingly, is independent of the tea1p–tea4p pathway. Conclusions These studies provide a direct demonstration for how MTs induce actin assembly at specific locations on the cell cortex and begin to identify a new pathway involved in this process. MT interactions with the cortex may be regulated by cortical attachment sites. These findings highlight the crosstalk between cell shape, polarity mechanisms, and MTs responsible for cell morphogenesis.

Summary Background During endocytosis in yeast, a choreographed series of discrete local events at the plasma membrane lead to a rapid burst of actin polymerization and then internalization of an endocytic vesicle. What initiates Arp2/3-dependent actin polymerization in this process is not well understood. Results The Schizosaccharomyces pombe WISH/DIP/SPIN90 orthologue dip1p is an actin patch protein that regulates the temporal sequence of endocytic events. dip1Δ mutants exhibit a novel phenotype in which early events such as WASp localization occur normally, but arrival of Arp2/3, actin polymerization and subsequent steps are delayed and occur with apparently random timing. In studying this mutant, we demonstrate that positive feedback loops of WASp, rapid actin assembly, and Arp2/3 contribute to switch-like behavior that initiates actin polymerization. In the absence of dip1p, a subset of patches is activated concurrently with the “touch” of a neighboring endocytic vesicle. Conclusions These studies reveal a switch-like mechanism responsible for the initiation of actin assembly during endocytosis. This switch may be activated in at least two ways, through a dip1p-dependent mechanism and through contact with another endocytic vesicle.

Summary T and B lymphocytes communicate by forming immunological synapses with antigen-presenting target cells. These highly dynamic contacts are characterized by continuous cytoskeletal remodeling events, which not only structure the interface but also exert a considerable amount of mechanical force. In recent years, it has become increasingly clear that synaptic forces influence information transfer both into and out of the lymphocyte. Here, we review our current understanding of synapse mechanics, focusing on its role as an avenue for intercellular communication.

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