scholarly journals How we discovered fluorescent speckle microscopy

2011 ◽  
Vol 22 (21) ◽  
pp. 3940-3942 ◽  
Author(s):  
E. D. Salmon ◽  
Clare M. Waterman
Keyword(s):  
Epithelial Cells ◽  
Fluorescence Intensity ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Living Cells ◽  
Variable Intensity ◽  
Macromolecular Assemblies ◽  
Dynamic Assembly ◽  
Fluorescence Images

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ∼10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual “speckled” appearance—there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

scholarly journals Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Charlotte F Kelley ◽  
Thomas Litschel ◽  
Stephanie Schumacher ◽  
Dirk Dedden ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Network Formation ◽  
Focal Adhesions ◽  
Membrane Composition ◽  
Macromolecular Assemblies ◽  
In Vitro Reconstitution ◽  
Membrane Bound ◽  
Underlying Mechanisms ◽  
Higher Order Networks ◽  
Made In

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

Locomotory invasion of human cervical epithelium and avian fibroblasts by HeLa cells in vitro

1982 ◽  
Vol 57 (1) ◽  
pp. 293-314 ◽  
Author(s):  
E.M. Stephenson
Keyword(s):  
Epithelial Cells ◽  
Hela Cells ◽  
Time Lapse ◽  
Tumour Cells ◽  
Contact Inhibition ◽  
Type I ◽  
Original Direction ◽  
Epithelial Sheet ◽  
Invasion Index

The locomotory invasive ability of HeLa cells was tested against: (a) embryonic chick heart fibroblasts (CHF); and (b) normal epithelial cells from human cervix (HCE) in explant confrontations. Data for analyses were obtained from replicate cultures fixed 24 h after junction and from 24-h time-lapse films. The mean invasion index for HeLa versus CHF did not indicate significant obstruction but analyses of hourly radial advance and orientation frequencies showed that obstruction eventually developed as postjunctional incubation time increased. Early contacts between HeLa and CHF demonstrated non-reciprocity of type I contact inhibition of locomotion by the tumour cells, which continued moving in their original direction to underlap contact-inhibited fibroblasts and eventually to occupy spaces vacated by them. When CHF population density increased and free space diminished, HeLa cells displayed directional and probably substrate-dependent contact inhibition. The high invasion index of HeLa versus HCE was largely due to occupation of previous HCE territory by tumour cells and only occasionally to actual infiltration of the epithelial sheet. After contact with HeLa, ruffling substrate-adherent marginal epithelial cells displayed contractile, type I contact inhibition of locomotion. After orientation changes, they gradually retreated. Against HCE, HeLa cells exhibited non-reciprocity of type I contact inhibition and continued radially forward, following the retreating epithelial margin. They did not move onto exposed upper surfaces of epithelial cells and did not underlap marginal cells firmly adherent to the substratum. Invasion of the epithelial sheet was seen only when initial access beneath a cell with a non-adherent margin was available. The contact relationships of isolated invading HeLa cells with their epithelial neighbours suggested successive non-reciprocal contact inhibition reactions.

scholarly journals Imaging cell biology in live animals: Ready for prime time

2013 ◽  
Vol 201 (7) ◽  
pp. 969-979 ◽  
Author(s):  
Roberto Weigert ◽  
Natalie Porat-Shliom ◽  
Panomwat Amornphimoltham
Keyword(s):  
Cell Biology ◽  
Cancer Biology ◽  
In Vitro Model ◽  
Time Lapse ◽  
Living Cells ◽  
Model Systems ◽  
Prime Time ◽  
Vitro Model

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.

scholarly journals Viscoelasticity of basal plasma membranes and cortices derived from MDCK II cells

2021 ◽  
Author(s):  
Andreas Janshoff
Keyword(s):  
Mechanical Properties ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Scaling Law ◽  
Focal Adhesions ◽  
Living Cells ◽  
The Body ◽  
Basolateral Membranes ◽  
Apical Side

In mature epithelial cells, however, cells adhere to one another through tight junctions, adherens junctions and desmosomes thereby displaying a pronounced apical-basal polarity. In vivo, the apical membrane has a larger surface area and faces the outer surface of the body or the lumen of internal cavities, whereas the basolateral membrane is oriented on the side away from the lumen and forms focal adhesions with the extracellular matrix. The mechanical properties of cells are largely determined by the architecture and dynamics of their viscoelastic cortex, which consists of a contractile, cross-linked actin mesh attached to the plasma membrane via linker proteins. Measuring the mechanical properties of adherent, polarized epithelial cells is usually limited to the upper, i.e., apical side of the cells due to their accessibility on culture dishes. Moreover, contributions from the cell interior comprising various filament types, organelles, and the crowded cytoplasm usually impede examination of the cortex alone. Here, we investigate the viscoelastic properties of basolateral membranes derived from polarized MDCK II epithelia in response to external deformation and compare them to living cells probed at the apical side. Therefore, we grew MDCK II cells on porous surfaces to confluency and removed the upper cell body by sandwich cleavage. The free-standing, defoliated cortices were subject to force indentation and relaxation experiments permitting a precise assessment of cortical viscoelasticity. A new theoretical framework to describe the force cycles is developed and applied to obtain the time-dependent area compressibility modulus of cell cortices from adherent cells. Compared to the viscoelastic response of living cells the basolateral membranes are substantially less fluid and stiffer but obey to the same universal scaling law if excess area is taken into account.

scholarly journals Counting the platelets: a robust and sensitive quantification method for thrombus formation

2016 ◽  
Vol 115 (06) ◽  
pp. 1178-1190 ◽  
Author(s):  
Tomas Lindahl ◽  
Lars Faxälv ◽  
Kjersti Claesson
Keyword(s):  
Platelet Count ◽  
Fluorescence Intensity ◽  
Thrombus Formation ◽  
Time Lapse ◽  
Flow Chamber ◽  
Volume Estimation ◽  
Research Groups ◽  
Study Objective ◽  
The Stability

SummaryFlow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity among the flow chamber systems currently used, and also in the methods used for quantifying the thrombus development. It was the study objective to evaluate a new method for analysis and quantification of platelet thrombus formation that can facilitate comparison of results between research groups. Whole blood was drawn over a collagen patch in commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets were fluorescently labelled and z-stack time-lapse images were captured during thrombus formation. Images were processed in a Python script in which the number of platelets and their respective x-, yand z-positions were obtained. For comparison with existing methods the platelets were also labelled and quantified using fluorescence intensity and thrombus volume estimations by confocal microscopy. The presented method was found less sensitive to microscope and image adjustments and provides more details on thrombus development dynamics than the methods for measuring fluorescence intensity and thrombus volume estimation. The platelet count method produced comparable results with commercial and PDMS flow chambers, and could also obtain information regarding the stability of each detected platelet in the thrombus. In conclusion, quantification of thrombus formation by platelet count is a sensitive and robust method that enables measurement of platelet accumulation and platelet stability in an absolute scale that could be used for comparisons between research groups.

scholarly journals Spatial distribution and functional significance of activated vinculin in living cells

2005 ◽  
Vol 169 (3) ◽  
pp. 459-470 ◽  
Author(s):  
Hui Chen ◽  
Daniel M. Cohen ◽  
Dilshad M. Choudhury ◽  
Noriyuki Kioka ◽  
Susan W. Craig
Keyword(s):  
Conformational Change ◽  
Direct Evidence ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Binding ◽  
Binding Conformation ◽  
Time Lapse Imaging

Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin β by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.

scholarly journals Regulation of Myosin II Dynamics by Phosphorylation and Dephosphorylation of Its Light Chain in Epithelial Cells

2007 ◽  
Vol 18 (2) ◽  
pp. 605-616 ◽  
Author(s):  
Toshiyuki Watanabe ◽  
Hiroshi Hosoya ◽  
Shigenobu Yonemura
Keyword(s):  
Epithelial Cells ◽  
Light Chain ◽  
Fluorescent Protein ◽  
Myosin Ii ◽  
Time Lapse ◽  
Stress Fibers ◽  
Interacting Protein ◽  
Cytoskeleton Organization

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.

scholarly journals On the Relationship Between EB-3 Profiles and Microtubules Growth in Cultured Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Arshat Urazbaev ◽  
Anara Serikbaeva ◽  
Anna Tvorogova ◽  
Azamat Dusenbayev ◽  
Sholpan Kauanova ◽  
...  
Keyword(s):  
Growth Velocity ◽  
Cultured Cells ◽  
Time Lapse ◽  
Living Cells ◽  
Time Intervals ◽  
Mean Values ◽  
Microtubule Growth ◽  
Dynamic Structures

Microtubules are dynamic structures undergoing rapid growth and shrinkage in living cells and in vitro. The growth of microtubules in vitro was analyzed with subpixel precision (Maurer et al., Current Biology, 2014, 24 (4), 372–384); however, to what extent these results could be applied for microtubules growing in vivo remains largely unknown. Particularly, the question is whether microtubule growth velocity in cells could be sufficiently approximated by a Gaussian distribution or its variability requires a more sophisticated description? Addressing this question, we used time-lapse microscopy and mathematical modeling, and we analyzed EB-3 comets forming on microtubules of cultured cells with subpixel precision. Parameters of comets (shape, form, and velocity) were used as topological characteristics of 3D voxel objects. Using regression analysis, we determined the real positions of the microtubule tips in time-lapse sequences. By exponential decay fitting of the restored comet intensity profile, we found that in vivo EB-3 rapidly exchanges on growing microtubule ends with a decoration time ∼ 2 s. We next developed the model showing that the best correlation between comet length and microtubule end growth velocity is at time intervals close to the decoration time. In the cells, EB comet length positively correlates with microtubule growth velocity in preceding time intervals, while demonstrating no correlation in subsequent time intervals. Correlation between comet length and instantaneous growth velocity of microtubules remains under nocodazole treatment when mean values of both parameters decrease. Our data show that the growth of microtubules in living cells is well-approximated by a constant velocity with large stochastic fluctuations.

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