Effie Bastounis

Both contractile axial and lateral traction force dynamics drive amoeboid cell motility

During chemotactic movement, D. discoideum exhibits step-wise amoeboid motility driven by both contractile axial forces and lateral forces.

Three-Dimensional Balance of Cortical Tension and Axial Contractility Enables Fast Amoeboid Migration

Fast amoeboid migration requires cells to apply mechanical forces on their surroundings via transient adhesions. However, the role these forces play in controlling cell migration speed remains largely unknown. We used three-dimensional force microscopy to measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examined wild-type cells as well as mutants with defects in contractility, internal F-actin crosslinking, and cortical integrity. We showed that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. We found that the migration speed increases when axial contractility overcomes cortical tension to produce the cell shape changes needed for locomotion. We demonstrated that the three-dimensional pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate without adhering to it, and which may be relevant for amoeboid migration in complex three-dimensional environments.

The Scar/WAVE complex is necessary for proper regulation of traction stresses during amoeboid motility

A combination of traction force and F-actin measurements shows that cells lacking either of the SCAR/WAVE complex proteins SCAR and PIR121 exhibit an altered cell motility cycle and spatiotemporal distribution of tractions stresses, which correlate in magnitude with F-actin levels.

The role of carotid plaque echogenicity in baroreflex sensitivity.

OBJECTIVE The baroreflex sensitivity is impaired in patients with carotid atherosclerosis. The purpose of our study was to assess the impact of carotid plaque echogenicity on the baroreflex function in patients with significant carotid atherosclerosis, who have not undergone carotid surgery. METHOD Spontaneous baroreflex sensitivity (sBRS) was estimated in 45 patients with at least a severe carotid stenosis (70%-99%). sBRS calculation was performed noninvasively, with the spontaneous sequence method, based on indirectly estimated central blood pressures from radial recordings. This method failed in three patients due to poor-quality recordings, and eventually 42 patients were evaluated. After carotid duplex examination, carotid plaque echogenicity was graded from 1 to 4 according to Gray-Weale classification and the patients were divided into two groups: the echolucent group (grades 1 and 2) and the echogenic group (grades 3 and 4). RESULTS Sixteen patients (38%) and 26 patients (62%) were included in the echolucent and echogenic group, respectively. Diabetes mellitus was observed more frequently among echolucent plaques (χ(2) = 8.0; P < .004), while those plaques were also more commonly symptomatic compared with echogenic atheromas (χ(2) = 8.5; P < .003). Systolic arterial pressure, diastolic arterial pressure, and heart rate were similar in the two groups. Nevertheless, the mean value of baroreflex sensitivity was found to be significantly lower in the echogenic group (2.96 ms/mm Hg) compared with the echolucent one (5.0 ms/mm Hg), (F [1, 42] = 10.1; P < .003). CONCLUSIONS These findings suggest that echogenic plaques are associated with reduced baroreflex function compared with echolucent ones. Further investigation is warranted to define whether such an sBRS impairment could be responsible for cardiovascular morbidity associated with echogenic plaques.

Cooperative cell motility during tandem locomotion of amoeboid cells

Tandem pairs of Dictyostelium cells migrate synchronously with an ~54-s time delay between the formation of their frontal protrusions. Each cell establishes two active adhesions, with the trailing cell reusing the location of the adhesions of the leading cell. This coordinated motility is mechanically driven and aided by cell–cell adhesions.

Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion

Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during migration.

Distribution of traction forces associated with shape changes during amoeboid cell migration

Amoeboid motility results from the cyclic repetition of shape changes leading to periodic oscillations of the cell length (motility cycle). We analyze the dominant modes of shape change and their association to the traction forces exerted on the substrate using Principal Component Analysis (PCA) of time-lapse measurements of cell shape and traction forces in migrating Dictyostelium cells. Using wild-type cells (wt) as reference, we investigated Myosin II activity by studying Myosin II heavy chain null cells (mhcA-) and Myosin II essential light chain null cells (mlcE-). We found that wt, mlcE-and mhcA- cells utilize similar modes of shape changes during their motility cycle, although these shape changes are implemented at a slower pace in Myosin II null mutants. The number of dominant modes of shape changes is surprisingly few with only four modes accounting for 75% of the variance in all cases. The three principal shape modes are dilation/elongation, bending, and bulging of the front/back. The second mode, resulting from sideways protrusion/retraction, is associated to lateral asymmetries in the cell traction forces, and is significantly less important in mhcA- cells. These results indicate that the mechanical cycle of traction stresses and cell shape changes remains remarkably similar for all cell lines but is slowed down when myosin function is lost, probably due to a reduced control on the spatial organization of the traction stresses.

Alterations of baroreflex sensitivity after carotid endarterectomy according to the preoperative carotid plaque echogenicity

OBJECTIVE Baroreflex sensitivity is lower in patients with echogenic carotid plaques compared with patients with echolucent ones. The purpose of our study was to compare the baroreflex function after carotid endarterectomy (CEA) between patients with different plaque echogenicity. METHOD Spontaneous baroreflex sensitivity (sBRS), heart rate, and systolic and diastolic arterial pressure were calculated in 51 patients with a severe carotid stenosis (70%-99%) 24 hours before CEA, as well as 24 and 48 hours after CEA. Carotid plaque echogenicity was graded from 1 to 4 according to Gray-Weale classification, after duplex examination, and the patients were divided into two groups: the echolucent (grade 1 or 2) and the echogenic (grade 3 or 4). RESULTS The postoperative mean systolic arterial pressure values in all 51 patients at 24 and 48 hours (143.2 and 135.5 mm Hg, respectively) were found to be significantly increased compared with the preoperative value (132.5 mm Hg; x2=32, P<.001). Mean sBRS value, in all patients, was significantly reduced postoperatively to 2.1 ms mm Hg(-1), from the mean preoperative value, 3.7 ms mm Hg(-1), independently of plaque echogenicity. Twenty patients (39%) were included in the echolucent group and 31 (61%) in the echogenic. The two groups had significant differences in two parameters: the rate of diabetes mellitus and the rate of symptomatic plaques. After adjusting the two groups for these differences, we found that the preoperative difference in sBRS between the two groups (F[1,51]=11, P<.003) was eliminated 24 and 48 hours after CEA (F[1,51]=.007, P<.9 and F[1,51]=.4, P<.5 for 24 and 48 hours, respectively). CONCLUSIONS Before the removal of carotid atheroma, baroreflex sensitivity, which is a well established cardiovascular risk factor, seems to be affected by carotid plaque echogenicity. However, CEA has as a result a similar baroreflex response in all patients, regardless of plaque echogenicity, implying no association of plaque morphology and postoperative baroreflex sensitivity.

The Role of the Scar/WAVE Complex in the Mechanics of Cell Migration

Cell motility is integral to a wide spectrum of biological phenomena. It requires the spatiotemporal coordination of underlying biochemical processes, resulting in cyclic shape changes associated with mechanical events (the motility cycle). A major driving force of cell migration is the dendritic polymerization of actin at the leading edge, regulated through the nucleation activity of the Arp2/3 complex, activated by the Scar/WAVE complex. Our aim is to understand the effect of the different components of the Scar/WAVE complex in the mechanics and in particular the motility cycle of migrating cells.For this purpose, we acquired time-lapse recordings of cell shape and traction forces of Dictyostelium cells migrating on deformable substrates. We compared results for wild-type cells and cells lacking the Scar/WAVE complex proteins PIR121 (Sra-1/CYFIP/GEX-2) (pirA-) and SCAR (scrA-). We find that mutantcells move slower than wild-type, while maintaining the overall characteristics of the mechanical interaction with the substrate, attaching at front and back and contracting inwards. Although the distribution of applied forces is unchanged, their magnitude is lower than in wild-type for scrA- cells and higher for pirA- cells. This correlates with the F-actin content of the different cell lines corroborating a role for F-actin in determining the level of the traction stresses.In pirA- cells regularity of the motility cycle (quasiperiodic repetition of shape changes and strain energy deposited) seems to be reduced compared to wild-type. This suggests that proper regulation of the Scar/WAVE complex and its role in F-actin turnover is essential for amoeboid motility.

Spatiotemporal Analysis of Traction Work Produced by Migrating Amoeboid Cells

Amoeboid cell motility is a complicated process requiring the regulated activity and localization of many molecules and resulting in the cyclic repetition of a relatively small repertoire of shape changes. These changes are driven by the traction work produced by the cell, which can be estimated by measuring the forces and displacements exerted by the cells on their substrate during migration. We have developed and applied a novel implementation of Principal Component Analysis to identify and sort out the most important shape changes in terms of traction work produced by chemotaxing Dictyostelium cells. For this purpose, we acquired time-lapse recordings of cell shape and traction forces of Dictyostelium cells migrating on deformable substrates. Using wild-type cells as reference, we investigated the effect of altering myosin II activity by studying myosin II null cells and essential light chain null cells. Our results indicate that the spatio-temporal variation of the traction work produced by Dictyostelium cells can be described with a reduced number of modes. In fact, only four modes are needed to account for 65% of the traction work exerted by all cells lines studied. Furthermore, the first mode alone accounts for more than 40% of the traction work. Spatially, this mode consists of the attachment of the cell predominantly at two areas at front and back, contracting towards the center of the cell. The time evolution of this mode is approximately periodic and coincides with the time evolution of cell length. Each one of the remaining modes accounts for less that 10% of the traction work. Their temporal and spatial organization is less clear, suggesting that the cell performs a traction work cycle composed of a repetitive sequence of steps over which random fluctuations are imposed.