Lamellipodia, stress fibers and the actin cortex of motile cells: Their structure and dynamic interaction studied by video DIC microscopy and correlated Electron Microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 428-429
Author(s):  
Julian P. Heath ◽  
Bruce F. Holifield
Keyword(s):  
Electron Microscopy ◽  
Ccd Camera ◽  
Time Lapse ◽  
Stress Fibers ◽  
Cortical Actin ◽  
Functional Relationships ◽  
Correlated Electron ◽  
Actin Cortex ◽  
Motile Cells ◽  
Contrast Enhanced

We have used high resolution video differential interference contrast (VDIC) light microscopy and correlated electron microscopy to examine the spatial and functional relationships between actin structures in the lamellipodia, adhesion plaques, stress fibers and the dorsal cortical actin microfilament sheath (DCMS) of cultured motile fibroblasts. Fibroblasts were examined on a Zeiss Axiophot using a 1.3NA 100x plan objective and 1.4 NA condenser and imaged with a Dage CCD camera. Contrast-enhanced images were recorded on a Panasonic S-VHS time-lapse recorder.

scholarly journals Generation of stress fibers through myosin-driven re-organization of the actin cortex

2020 ◽  
Author(s):  
JI Lehtimäki ◽  
EK Rajakylä ◽  
S Tojkander ◽  
P Lappalainen
Keyword(s):  
De Novo ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Cellular Processes ◽  
Actin Cortex ◽  
Muscle Myosin ◽  
Subsequent Stabilization

SummaryContractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, are generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex, and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.

scholarly journals Generation of stress fibers through myosin-driven reorganization of the actin cortex

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jaakko I Lehtimäki ◽  
Eeva Kaisa Rajakylä ◽  
Sari Tojkander ◽  
Pekka Lappalainen
Keyword(s):  
De Novo ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Cellular Processes ◽  
Actin Cortex ◽  
Muscle Myosin ◽  
Subsequent Stabilization

Contractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, are generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.

scholarly journals Reorganization of actin filament bundles in living fibroblasts.

1984 ◽  
Vol 99 (4) ◽  
pp. 1478-1485 ◽  
Author(s):  
Y L Wang
Keyword(s):  
Fluorescence Intensity ◽  
Cell Movement ◽  
Leading Edge ◽  
Time Lapse ◽  
Stress Fibers ◽  
Initial Structure ◽  
Actin Bundles ◽  
Motile Cells ◽  
Filament Bundles ◽  
As If

We investigated how actin bundles assemble, disassemble, and reorganize during cell movement. Living chick embryonic fibroblasts were microinjected with actin molecules that had been fluorescently labeled with tetramethylrhodamine. We found that the fluorescent analogue of actin can be used successfully by both existing and newly formed cellular structures. Using time-lapse photography coupled to image-intensified fluorescence microscopy, we were able to detect various patterns of reorganization in motile cells. Assembly of stress fibers occurred near both the leading and the trailing ends of the cell. The initial structure appeared as discrete spots that subsequently extended into stress fibers. The extension occurred unidirectionally. The site of initiation near the leading edge remained stationary relative to the substrate during subsequent cell advancement. However, the orientation of the fiber could change according to the direction of cell movement. In addition, existing stress fibers could merge or fragment. The shortening of stress fibers can occur from either end of the fiber. Shortening from the proximal end (centrifugal shortening) was accompanied by a decrease in fluorescence intensity, as if the bundle were disassembling, and usually led to the total disappearance of the bundle. Shortening from the distal end (centripetal shortening), on the other hand, is usually accompanied by an increase in fluorescence intensity at the distal end of the bundle, as if this end had pulled loose from its attachment and retracted toward the center of the cell. Besides stress fibers, arc-like actin bundles have also been detected in spreading cells. These observations can explain how the organization of actin bundles coordinates with cell movement, and how stress fibers reach a highly regular pattern in static cells.

Zero-loss omega-filtered REM and RHEED on the new Zeiss 912

1991 ◽  
Vol 49 ◽  
pp. 616-617
Author(s):  
J.C.H. Spence ◽  
J. Mayer
Keyword(s):  
Electron Microscopy ◽  
Materials Science ◽  
Surface States ◽  
Ccd Camera ◽  
Dark Field ◽  
Ion Pump ◽  
Magnetic Imaging ◽  
Reflection Electron Microscopy ◽  
Diffraction Patterns ◽  
Large Background

The Zeiss 912 is a new fully digital, side-entry, 120 Kv TEM/STEM instrument for materials science, fitted with an omega magnetic imaging energy filter. Pumping is by turbopump and ion pump. The magnetic imaging filter allows energy-filtered images or diffraction patterns to be recorded without scanning using efficient parallel (area) detection. The energy loss intensity distribution may also be displayed on the screen, and recorded by scanning it over the PMT supplied. If a CCD camera is fitted and suitable new software developed, “parallel ELS” recording results. For large fields of view, filtered images can be recorded much more efficiently than by Scanning Reflection Electron Microscopy, and the large background of inelastic scattering removed. We have therefore evaluated the 912 for REM and RHEED applications. Causes of streaking and resonance in RHEED patterns are being studied, and a more quantitative analysis of CBRED patterns may be possible. Dark field band-gap REM imaging of surface states may also be possible.

High-voltage Electron Microscopy of astroglial cell whole mounts

1986 ◽  
Vol 44 ◽  
pp. 316-317
Author(s):  
J.N. Turner ◽  
W.G. Shain ◽  
V. Madelian ◽  
R.A. Grassucci ◽  
D.L. Forman
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Time Lapse ◽  
Astroglial Cells ◽  
High Voltage Electron Microscopy ◽  
Rat Glioma ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Nervous System Function ◽  
Beta Adrenergic Agonist

Homogeneous cultures of astroglial cells have proved useful for studying biochemical, pharmacological, and toxicological responses of astrocytes to effectors of central nervous system function. LRM 55 astroglial cells, which were derived from a rat glioma and maintained in continuous culture, exhibit a number of astrocyte properties (1-3). Stimulation of LRM 55s and astrocytes in primary cell cultures with the beta-adrenergic agonist isoproterenol results in rapid changes of morphology. Studies with time lapse video light microscopy (VLM) and high-voltage electron microscopy (HVEM) have been correlated to changes in intracellular levels of c-AMP. This report emphasizes the HVEM results.

Three-dimensional organization and functional relationships of endocytotic compartments in pig intestinal epithelial cells

1992 ◽  
Vol 50 (1) ◽  
pp. 576-577
Author(s):  
Julian P. Heath ◽  
Buford L. Nichols ◽  
László G. Kömüves
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Intestinal Epithelial ◽  
Cell Surfaces ◽  
Basal Surface ◽  
Functional Relationships

The newborn pig intestine is adapted for the rapid and efficient absorption of nutrients from colostrum. In enterocytes, colostral proteins are taken up into an apical endocytotic complex of channels that transports them to target organelles or to the basal surface for release into the circulation. The apical endocytotic complex of tubules and vesicles clearly is a major intersection in the routes taken by vesicles trafficking to and from the Golgi, lysosomes, and the apical and basolateral cell surfaces.Jejunal tissues were taken from piglets suckled for up to 6 hours and prepared for electron microscopy and immunocytochemistry as previously described.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

Motility of the Limulus blood cell

1979 ◽  
Vol 37 (1) ◽  
pp. 169-180
Author(s):  
P.B. Armstrong
Keyword(s):  
Tissue Culture ◽  
Time Lapse ◽  
Coelomic Fluid ◽  
Cellular Motility ◽  
Paraffin Oil ◽  
Motile Cells ◽  
Direct Microscopic Examination ◽  
High Concentrations ◽  
Glass Surfaces

The sole cell type (the amoebocyte) found in the coelomic fluid of the horseshoe crab, Limulus polyphemus can be stimulated to become motile by extravasation or trauma. Motility was studied using time-lapse microcinematography and direct microscopic examination of cells in tissue culture and in gill leaflets isolated from young animals. Phase-contrast and Nomarski differential-interference contrast optics were employed. Both in culture and in the gills, motile cells showed 2 interconvertible morphological types: the contracted cell, which was compact and rounded and had a relatively small area of contact with the substratum, and a flattened from with a larger area of contact. In both morphological types, motility involved the protrusion of hyaline pseudopods followed by flow of granular endoplasm forward in the pseudoplod. Cellular motility in vivo (in the gill leaflet) was morphologically identical to that displayed in tissue culture. In culture, motility was unaffected by the nature of the substratum: cells were indistinguishable on fluid (paraffin oil) or solid (glass) substrata or on hydrophobic (paraffin oil, siliconized glass) or hydrophilic (clean glass) surfaces. Cells migrated and spread on agar surfaces. Cell motility was unaffected by high concentrations (100 micrograms/ml) of the microtubule-depolymerizing agent colcemid and was abolished by cytochalasin B at 1 microgram/ml.

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