No abstract

ETOC: Lgl1 interacts directly with NMII-A, inhibiting its filament assembly. It excludes NMII-A from the cell leading edge. Depletion of Lgl1 affects the size and number of focal adhesions, cell polarity, and the rate of migrating cells. These results indicate that Lgl1 regulates the polarity of migrating cells by controlling NMII-A filament assembly.

Many signaling pathways regulate the function of the cellular cytoskeleton. Yet we know very little about the proteins involved in the cross-talk between the signaling and the cytoskeletal systems. Here we show that myosin II-B, an important cytoskeletal protein, resides in a complex with p21-activated kinase 1 (PAK1) and atypical protein kinase C (PKC) zeta (aPKC) and that the interaction between these proteins is EGF-dependent. We further show that PAK1 is involved in aPKC phosphorylation and that aPKC phosphorylates myosin II-B directly on a specific serine residue in an EGF-dependent manner. This latter phosphorylation is specific to isoform B of myosin II, and it leads to slower filament assembly of myosin II-B. Furthermore, a decrease in aPKC expression in the cells alters myosin II-B cellular organization. Our finding of a new signaling pathway involving PAK1, aPKC, and myosin II-B, which is implicated in myosin II-B filament assembly and cellular organization, provides an important link between the signaling system and cytoskeletal dynamics.

Non muscle myosin II (NMII) is a major motor protein present in all cell types. The three known vertebrate NMII isoforms share high sequence homology but play different cellular roles. The main difference in sequence resides in the C-terminal nonhelical tailpiece (tailpiece). In this study we demonstrate that the tailpiece is crucial for proper filament size, overcoming the intrinsic properties of the coiled-coil rod. Furthermore, we show that the tailpiece by itself determines the NMII filament structure in an isoform-specific manner, thus providing a possible mechanism by which each NMII isoform carries out its unique cellular functions. We further show that the tailpiece determines the cellular localization of NMII-A and NMII-B and is important for NMII-C role in focal adhesion complexes. We mapped NMII-C sites phosphorylated by protein kinase C and casein kinase II and showed that these phosphorylations affect its solubility properties and cellular localization. Thus phosphorylation fine-tunes the tailpiece effects on the coiled-coil rod, enabling dynamic regulation of NMII-C assembly. We thus show that the small tailpiece of NMII is a distinct domain playing a role in isoform-specific filament assembly and cellular functions. Non muscle myosin II (NMII)2 is a major motor protein present in all cell types participating in crucial processes, including cytokinesis, surface attachment, and cell movement (1-3). NMII units are hexamers of two long heavy chains with two pairs of light chains attached. NMII heavy chain is composed of a globular head containing the actin binding and force generating ATPase domains, followed by a large coiled-coil rod that terminates with a short non-helical tailpiece (tailpiece). To carry out its cellular functions, NMII assembles into dimers and higher order filaments by interactions of the coiled-coil rod (4). The assembly process is governed by electrostatic interactions between adjacent coiled-coil rods containing alternating charged regions with specific periodicity (5-9) and is enhanced by activation of the motor domain through regulatory light chain phosphorylation (10 -12). The charge periodicity also determines the register and orientation of each NMII hexamer in the filament. Additionally the C-terminal region of the coiled-coil rod contains a distinctive positively charged region and the assembly-competence domains that are crucial for proper filament assembly (5-9, 13).Three isoforms of NMII (termed NMII-A, NMII-B, and NMII-C) have been identified in mammals (14 -16). Although NMII isoforms share somewhat overlapping roles, each isoform has distinctive tissue distribution and specific functions. NMII-A is important for neural growth cone retraction (17,18) and is distributed to the front of migrating endothelial cells (19). While NMII-B participates in growth cone advancement (20) and was detected in the retracting tails of migrating endothelial cells (19). Furthermore NMII-A and NMII-B have an opposing effect on motility, since depletion of NMII-A leads to increased mot...

When cells of the bacterium Salmonella typhimurium are incubated with penicillin and lysed in a dilute buffer, flagellated cytoplasm-free envelopes are formed. When the envelopes are tethered to glass by their flagella and then energized, some of them spin. The direction of rotation of wild-type envelopes is exclusively counterclockwise (CCW). We perturbed this system by including in the lysis medium (and hence in the envelopes) the chemotaxis protein CheY. As a result, some of the envelopes rotated exclusively clockwise (CW). The fraction of envelopes that did so increased with the concentration of CheY; at a concentration of 48 FM (pH 8), all functional envelopes spun CW. The fraction also increased with the pH of the lysis medium in the range 6.6-8.4. The results were the same in the presence or absence of intracellular Ca2+.Reconstituted envelopes failed to respond to chemotactic stimuli. None of them changed the direction of their rotation. However, when the intracellular pH was lowered to 6.6 or below, envelopes that spun CW stopped rotating, while envelopes that spun CCW continued to rotate. This phenomenon was reversible. We conclude that CheY per se, without any additional free cytoplasmic mediators, interacts with a switch at the base of the flagellum to cause CW rotation.Bacterial chemotaxis is migration toward favorable chemicals (attractants) and away from unfavorable ones (repellents) (1). The unstimulated behavior of peritrichous bacteria is smooth swimming with occasional briefperiods of tumbling (2, 3). This unstimulated mode of swimming is the consequence of alternating flagellar rotation: smooth swimming results from counterclockwise (CCW) rotation and tumbling results from clockwise (CW) rotation. Attractants or repellents shift the rotation to CCW or CW bias (4) and thus cause smooth swimming or tumbling, respectively. The molecular mechanism that regulates the direction of this rotation is not known.For studying this regulation mechanism, an in vitro system consisting of functional cell envelopes, isolated from Escherichia coli and Salmonella typhimurium (5), was used. The internal content of these envelopes can be controlled and predetermined. The envelopes have intact cytoplasmic membrane and parts of the cell wall. They are essentially free of cytoplasm and contain instead the medium in which the bacteria, from which the envelopes were derived, were lysed. Due to the absence of cytoplasm, the flagella of the envelopes do not rotate unless an electron donor is added for respiration (5,6). This lack of rotation in the absence of an added energy source serves as a control, carried out for every envelope, for lack of cytoplasmic remnants in it.