Juliet Lee

Regulation of cell movement is mediated by stretch-activated calcium channels

Intracellular calcium regulates many of the molecular processes that are essential for cell movement. It is required for the production of actomyosin-based contractile forces, the regulation of the structure and dynamics of the actin cytoskeleton,, and the formation and disassembly of cell–substratum adhesions,. Calcium also serves as a second messenger in many biochemical signal-transduction pathways. However, despite the pivotal role of calcium in motile processes, it is not clear how calcium regulates overall cell movement. Here we show that transient increases in intracellular calcium, [Ca2+]i, during the locomotion of fish epithelial keratocytes, occur more frequently in cells that become temporarily ‘stuck’ to the substratum or when subjected to mechanical stretching. We find that calcium transients arise from the activation of stretch-activated calcium channels, which triggers an influx of extracellular calcium. In addition, the subsequent increase in [Ca2+]i is involved in detachment of the rear cell margin. Thus, we have defined a mechanism by which cells can detect and transduce mechanical forces into biochemical signals that can modulate locomotion.

How do cells move along surfaces

The movement of cells along surfaces is a complex phenomenon that consists of several interrelated processes, including cell-substratum adhesion, and extension and retraction of the cell edge, in which the actin cytoskeleton plays a crucial role. The past decade has seen increasingly detailed molecular-based investigations into cell motility, but it is still not known how molecular events are integrated to give cell movement. Molecular studies are now beginning to be linked to a more global concept of how whole cells move, and this combined approach promises to yield new insights into cell locomotion.

Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement.

Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA–) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE–) and mhcA– cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it.

Cyclic changes in keratocyte speed and traction stress arise from Ca2+-dependent regulation of cell adhesiveness

The activation of stretch-activated calcium channels (SACs) in keratocytes can induce spatially coordinated increases in traction stress that promote protrusion at the cell front, while simultaneously inducing retraction at the rear. To investigate how this occurs, we correlated calcium-induced changes in traction stress with alterations in cell speed and shape. Cyclic changes in these parameters were associated with each calcium transient. In addition, an inverse relationship was found between traction stress and cell speed, suggesting that alternating changes in adhesiveness were occurring at the rear. We investigated this further by inhibiting or inducing calcium transients and observing the effects on traction stress, cell speed and shape. Inhibition of calcium transients prevented retraction and led to a slow increase in traction stress. In addition, large aggregates of vinculin developed at the lateral rear edges of treated keratocytes, consistent with an increase in adhesiveness. Induction of a calcium transient resulted in a rapid retraction, involving both increased traction stress and adhesion disassembly at the rear. We also found that keratocytes exhibiting frequent transients generated larger traction stress and moved significantly faster than other cells. Together, these data suggest that calcium transients coordinate changes in adhesiveness with SAC-mediated cycles of mechano-chemical feedback.

Microscopic methods for measuring the elasticity of gel substrates for cell culture: microspheres, microindenters, and atomic force microscopy

In conjunction with surface chemistry, the mechanical properties of cell culture substrates provide important biological cues that affect cell behavior including growth, differentiation, spreading, and migration. The phenomenon has led to the increased use of biological and synthetic polymer-based flexible substrates in cell culture studies. However, widely used methods for measuring the Youngs modulus have proven difficult in the characterization of these materials, as they tend to be relatively thin, soft, hydrated, and tethered to glass substrates. Here we describe three methods that have been applied successfully to probe the flexibility of soft culture substrates.

HSPB1, actin filament dynamics, and aging cells

Investigations into the possible roles of human HSPB1 in aging have focused on its role as a molecular chaperone protecting partially folded or unfolded proteins, particularly during oxidative stress. A thorough analysis of potential roles of HSPB1 in aging cells has been hampered by a limited knowledge of its functions in living cells. Most studies have employed cell‐free extracts and purified proteins. For example, HSPB1 is known to bind actin in vitro, and this observation led to the hypothesis that HSPB1 regulates actin filament dynamics. In the study summarized herein, the role of HSPB1 in regulating actin filament dynamics was further investigated by using cultured human cells. These results show that HSPB1 and actin form a complex in vivo and that HSPB1 is important for cell motility. A model for HSPB1 as a regulator of actin filament dynamics is presented, and evidence from the literature on cytoskeletal alterations in aging cells is discussed.

A ground-based model to study the effects of weightlessness on lymphocytes

The mitogenic response of human lymphocytes was found to be markedly reduced in weightlessness conditions as compared to normal gravity. One possible explanation is that due to the non‐existent sedimentation in space the lymphocytes could not adhere and spread on a substratum. Thus, we investigated the effect of substratum adhesiveness on lymphocyte responsiveness by reducing and blocking cell adhesion with poly‐HEMA in a simple on‐ground system. Lymphocyte adhesiveness was assessed by measuring the proportion of non‐adhesive, slightly, and strongly adhesive 51Cr‐radiolabelled cells on uncoated and poly‐HEMA coated plastic. The amount of cell spreading on surfaces with varying adhesiveness was determined by measuring the area of cells. Cells grown on medium and thick poly‐HEMA films were rounded in shape. By contrast, on tissue culture plastic, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly‐HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon‐γ production was virtually nil when the cells were grown on the least adhesive substratum. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca2+]i) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca2+]i. We observed small, brief, Ca2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd3+) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.

The use of gelatin substrates for traction force microscopy in rapidly moving cells.

The study of traction forces generated by rapidly moving cells requires the use of substrates that are highly elastic because these cells typically generate weaker traction forces than slower moving cells. Gelatin substrates are soft enough to allow deformation by rapidly moving cells such as fish epidermal keratocytes and Dictyostelium discoideum amoebas. In addition, gelatin substrates are thin (approximately 30-40 microm) and transparent, allowing them to be used in combination with high-resolution calcium imaging. Importantly, the responsiveness of gelatin substrates allows changes in traction force generation to be detected within seconds, corresponding to the timescale of calcium transients. Here we describe the manufacture and application of gelatin substrates to study the role of mechanochemical signaling in the regulation of keratocyte movement. We show how patterns of traction force generation can be analyzed from a time series of traction vector maps, and how to interpret them in relation to cell movement. In addition, we discuss how the gelatin traction force assay is being used to study the mechanics of Dictyostelium cell motility, and future applications such as the study of neuronal path finding.

Reduced lymphocyte activation in space: role of cell-substratum interactions.

We investigated the effect of substratum adhesiveness on stimulated lymphocyte blastogenesis by reducing and blocking cell adhesion with poly (2-hydroxyethyl methacrylate) (poly-HEMA) in a simple on-ground system. Cells grown on medium-thick and thick poly-HEMA films were rounded in shape and displayed no signs of spreading. By contrast, on tissue culture plastic and very thin poly-HEMA films, they showed clear signs of spreading. The mitogenic response of lymphocytes grown on thick poly-HEMA films was reduced by up to 68% of the control (tissue culture plastic). Interferon-gamma production was near zero when the cells were grown on the least adhesive substratum. On uncoated plastic, activated lymphocytes subjected to high gravity (20g) exhibited an increased proliferation rate (40%) compared with 1g. By contrast, on poly-HEMA, high gravity did not improve lymphocyte responsiveness. These results show that activated lymphocytes need to anchor and spread prior to achieving an optimal proliferation response. We conclude that decreased lymphocyte adhesion could contribute to the depressed in vitro lymphocyte responsiveness found in the microgravity conditions of space flight.