Biomedical subjects

Stephanie L Gupton

Publications and source records attributed to Stephanie L Gupton.

5 recordsLinked to original sources

PubMed2006

Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration.

Cells exhibit a biphasic migration-velocity response to increasing adhesion strength, with fast migration occurring at intermediate extracellular matrix (ECM) concentration and slow migration occurring at low and high ECM concentration. A simple mechanical model has been proposed to explain this observation, in which too little adhesion does not provide sufficient traction whereas too much adhesion renders cells immobile. Here we characterize a phenotype for rapid cell migration, which in contrast to the previous model reveals a complex interdependence of subcellular systems that mediates optimal cell migration in response to increasing adhesion strength. The organization and activity of actin, myosin II, and focal adhesions (FAs) are spatially and temporally highly variable and do not exhibit a simple correlation with optimal motility rates. Furthermore, we can recapitulate rapid migration at a nonoptimal ECM concentration by manipulating myosin II activity. Thus, the interplay between actomyosin and FA dynamics results in a specific balance between adhesion and contraction, which induces maximal migration velocity.

Actins↗

PubMed2005

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

Actin Depolymerizing Factors↗

PubMed2004

Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy.

We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion. However, how they are orchestrated to produce directed cell movements is poorly understood. Our approach to data analysis allows us to examine their interdependence. Our maps confirm the previously described organization of flow into a lamellipodium and a lamellum, both exhibiting retrograde flow; and a convergence zone, where lamellum retrograde flow meets with slow anterograde flow of cortical F-actin at the ventral side of the cell body. The turnover maps show the well known actin polymerization at the leading edge, but also indicate that approximately 90% of the polymer disassembles at the lamellipodium-lamellum junction. Strong depolymerization is also found in the convergence zone, where meshwork contraction is prominent. To determine whether contraction and depolymerization are coupled events, we have treated cells with calyculin A, which is known to promote myosin activity. Stimulated contraction was accompanied by accelerated retrograde flow and increased depolymerization throughout the lamellum, whereas disassembly at the lamellipodium-lamellum junction remained unaffected. There appear to be two distinct depolymerization mechanisms, of which one depends directly on meshwork contraction.

Actins↗

PubMed2003

A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells.

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100-300mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons.

Animals↗

PubMed2002

Converging populations of f-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells.

BACKGROUND: In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, ), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown. Here, we test the hypothesis that MT polymer loss occurs in central cell regions and is induced by the convergence of actin retrograde and anterograde flow, which buckles and breaks associated MTs and promotes minus-end depolymerization. RESULTS: We characterized the effects of calyculin A and ML-7 on the movement of f-actin and MTs by multi-spectral fluorescence recovery after photobleaching (FRAP) and fluorescent speckle microscopy (FSM). Our studies show that these drugs affect the rate of f-actin and MT convergence and MT buckling in a central cell region we call the "convergence zone." Increases in f-actin convergence are associated with faster MT turnover and an increase in both MT breakage and minus-end depolymerization, but they have no effect on MT plus end dynamic instability. CONCLUSIONS: We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.

Actins↗