Dual-color total internal reflection fluorescence microscopy revealed that the N-terminal half of Srv2 (N-Srv2) directly catalyzes severing of cofilin-decorated actin filaments. N-Srv2 formed novel six-bladed structures resembling ninja throwing stars (shurikens), and N-Srv2 activities were critical for actin organization in vivo and were lethal in combination with Aip1.

Notch-view radiographs were obtained from 108 persons with anterior cruciate ligament (ACL) injuries (55 women, 53 men) and 186 with intact ACL (94 women, 92 men). Notch width, femur width, and notch width index were determined from each of the 294 radiographs. The notch was also categorized as either A-shaped or non-A-shaped. Intrarater and interrater reliability ranged from 0.82 to 0.99 for notch width and femur width, respectively. Reliability within and between raters for the classification of notch shape ranged from 0.80 to 1.0. Notch width was significantly influenced by a 10 degree change in knee angle when repeated radiographs were taken. Femur width was not affected by knee angle across this range. Analysis revealed a higher proportion of A-shaped notches among women than men. However, notch shape was not related to injury status. Results showed a smaller notch width and notch width index in ACL-injured patients regardless of notch shape or gender. A-shaped notches were smaller than non-A-shaped notches regardless of injury status or gender. Both notch width and notch width index were found to be significant indicators of ACL injury. Knowledge of the shape of the notch added no useful information in differentiating patients based on injury status. Thus, regardless of gender, individuals who possess smaller notch dimensions appear to be at greater risk of injury than individuals with larger notches.

Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assemblycompetent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (K d ~1.5μM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange.

Recent evidence has suggested that Srv2/CAP (cyclase-associated protein) has two distinct functional roles in regulating actin turnover, with its N-terminus enhancing cofilin-mediated severing of actin filaments and its C-terminus catalyzing actin monomer recycling. However, it has remained unclear to what degree these two activities are coordinated by being linked in one molecule, or whether they can function autonomously. To address this, we physically divided the protein into two separate halves, N-Srv2 and C-Srv2, and asked whether they are able to function in trans both in living cells and in reconstituted assays for F-actin turnover and actin-based motility. Remarkably, in F-actin turnover assays the stimulatory effects of N-Srv2 and C-Srv2 functioning in trans were quantitatively similar to those of intact full-length Srv2. Further, in bead motility assays and in vivo, the fragments again functioned in trans, although not with the full effectiveness of intact Srv2. From these data, we conclude that the functions of the two halves of Srv2/CAP are largely autonomous, although their linkage improves coordination of the two functions in specific settings, possibly explaining why the linkage is conserved across distant plant, animal, and fungal species.

Swarming on rigid surfaces requires movement of cells as individuals and as a group of cells. For the bacterium Proteus mirabilis, an individual cell can respond to a rigid surface by elongating and migrating over micrometer-scale distances. Cells can form groups of transiently aligned cells, and the collective population is capable of migrating over centimeter-scale distances. To address how P. mirabilis populations swarm on rigid surfaces, we asked whether cell elongation and single-cell motility are coupled to population migration. We first measured the relationship between agar concentration (a proxy for surface rigidity), single-cell phenotypes, and swarm colony phenotypes. We find that cell elongation and single-cell motility are coupled with population migration on low-percentage hard agar (1% to 2.5%) and become decoupled on high-percentage hard agar (>2.5%). Next, we evaluate how disruptions in lipopolysaccharide (LPS), specifically the O-antigen components, affect responses to hard agar. We find that LPS is not essential for elongation and motility of individual cells, as predicted, and instead functions to broaden the range of agar concentrations on which cell elongation and motility are coupled with population migration. These findings demonstrate that cell elongation and motility are coupled with population migration under a permissive range of surface conditions; increasing agar concentration is sufficient to decouple these behaviors. Since swarm colonies cover greater distances when these steps are coupled than when they are not, these findings suggest that collective interactions among P. mirabilis cells might be emerging as a colony expands outwards on rigid surfaces. IMPORTANCE How surfaces influence cell size, cell-cell interactions, and population migration for robust swarmers like P. mirabilis is not fully understood. Here, we have elucidated how cells change length along a spectrum of sizes that positively correlates with increases in agar concentration, regardless of population migration. Single-cell phenotypes can be decoupled from collective population migration simply by increasing agar concentration. A cell’s lipopolysaccharides function to broaden the range of agar conditions under which cell elongation and single-cell motility remain coupled with population migration. In eukaryotes, the physical environment, such as a surface matrix, can impact cell development, shape, and migration. These findings support the idea that rigid surfaces similarly act on swarming bacteria to impact cell shape, single-cell motility, and collective population migration.

Individual cells of the bacterium can elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarming. How cells regulate this dramatic morphological remodeling remains an open question. In this paper, we move forward the understanding of this regulation by demonstrating that requires the gene for swarmer cell elongation and subsequent swarm motility. The gene encodes a protein homologous to the dTDP-glucose 4,6-dehydratase protein of , which contributes to enterobacterial common antigen biosynthesis. Here, we characterize the gene in , demonstrating that it is required for the production of large lipopolysaccharide-linked moieties necessary for wild-type cell envelope integrity. We show that the absence of the gene induces several stress response pathways, including those controlled by the transcriptional regulators RpoS, CaiF, and RcsB. We further show that in -deficient cells, the suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to induce cell elongation and swarm motility. However, the loss of RcsB does not rescue cell envelope integrity defects and instead results in abnormally shaped cells, including cells producing more than two poles. We conclude that an RcsB-mediated response acts to suppress the emergence of shape defects in cell envelope-compromised cells, suggesting an additional role for RcsB in maintaining cell morphology under stress conditions. We further propose that the composition of the cell envelope acts as a checkpoint before cells initiate swarmer cell elongation and motility. swarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multifunctional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, the -dependent moieties provide a novel attractive target for potential antimicrobials.

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Organisms can alter morphology and behaviors in response to environmental stimuli such as mechanical forces exerted by surface conditions. The bacterium Proteus mirabilis responds to surface-based growth by enhancing cell length and degree of cell-cell interactions. Cells grow as approximately 2-micrometer rigid rods and independently swim in liquid. By contrast on hard agar surfaces, cells elongate up to 40-fold into snake-like cells that move as a collective group across the surface. Here we have elucidated that individual cell size and degree of cell-cell interactions increased across a continuous gradient that correlates with increasing agar density. We further demonstrate that interactions between the lipopolysaccharide (LPS) component of the outer membrane and the immediate local environment modified these responses by reducing agar-associated barriers to motility. Loss of LPS structures corresponded with increased cell elongation on any given surface. These micrometer-scale changes to cell shape and collective interactions corresponded with centimeter-scale changes in the overall visible structure of the swarm colony. It is well-appreciated in eukaryotes that mechanical forces impact cell shape and migration. Here we propose that bacteria can also dynamically respond to the mechanical forces of surface conditions by altering cell shape, individual motility, and collective migration. IntroductionEukaryotic and prokaryotic cells undergo alterations, including changes in cell morphology and collective behaviors, in response to interactions with the physical environment. For example, robust swarmers such as the human pathogens Proteus mirabilis and Vibrio parahaemolyticus (1) can swim as short, rigid rod-shaped cells in liquid and through low-density (0.3%) agar. On low-wetness and highdensity agar (0.75% to 2.5%), these bacteria elongate dramatically and move as a collective group on top of the surface. By comparison, the swarm motility of temperate swarmers such as Escherichia coli or Salmonella enterica is generally restricted to low-density agar (< 0.7%) or high wetness Eiken agar (reviewed in (2, 3)). For all of the aforementioned bacteria, flagella power the motility in liquid and on surfaces. Furthermore, a transition from swimming to swarming coincides with a series of large-scale transcriptional changes triggered by contact between flagella and a surface (Figure 1) (4-12).Here we utilize P. mirabilis as a tractable model for exploring how morphology and cell-cell interactions respond to the surface. P. mirabilis cells are approximately 2-µm long, rigid, and rod-shaped in liquid. Such cells swim independently, resulting in a visible uniform haze to the structure of the swim colony. Upon contact with a hard surface (Figure 1), these rigid, rod-shaped cells can elongate up to 40-fold into a flexible, snake-like, hyper-flagellated swarmer cell (13,14) that has a distinctive gene expression profile (15). P. mirabilis swarmer cells are thought to bundle their flagella to facilitate cooperative swarm motil...