Temporal and Spatial Dynamics of the Actin-Based Cytoskeleton

1990 ◽  
Vol 48 (2) ◽  
pp. 546-546
Author(s):  
D. Lansing Taylor
Keyword(s):  
Spatial Dynamics ◽  
Time Lapse ◽  
Living Cells ◽  
Biomedical Sciences ◽  
3T3 Cells ◽  
Cellular Functions ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Temporal And Spatial ◽  
Time Lapse Imaging

There has been a renaissance and revolution in the use of light microscopy in the biomedical sciences. The renaissance has been due to the importance of studying the temporal and spatial dynamics of ions, metabolites and macromolecules in living cells and tissues. The revolution has been due to the integration of developments in molecular biology, fluorescent probe chemistry, machine vision, and imaging technology. It is now possible to use the living cell as a microcuvette and to explore the chemical and molecular dynamics responsible for cellular functions.We have been investigating the formation, transport and contraction of stress fibers in Swiss 3T3 cells. Fluorescent analogs of actin, myosin, vinculin and profilin have been investigated in serum deprived cells before, during and after stimulation with thrombin. The activities of these components of the actin-based cytoskeleton have been quantified using time-lapse imaging, fluorescence redistribution after photobleaching, video-enhanced contrast and reflection interference contrast microscopy.

scholarly journals Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells.

1987 ◽  
Vol 105 (4) ◽  
pp. 1613-1622 ◽  
Author(s):  
R DeBiasio ◽  
G R Bright ◽  
L A Ernst ◽  
A S Waggoner ◽  
D L Taylor
Keyword(s):  
Fluorescent Probes ◽  
Near Infrared ◽  
Spatial Information ◽  
Spatial Dynamics ◽  
Stress Fibers ◽  
Chemical Components ◽  
3T3 Cells ◽  
Cellular Functions ◽  
Temporal And Spatial ◽  
Swiss 3T3

Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.

scholarly journals The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

2001 ◽  
Vol 154 (6) ◽  
pp. 1135-1146 ◽  
Author(s):  
Aime A. Levesque ◽  
Duane A. Compton
Keyword(s):  
Cultured Cells ◽  
Time Lapse ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Poles ◽  
Ejection Force ◽  
Chromosome Congression ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.

scholarly journals EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

2017 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Cellular Functions ◽  
Binding Ability ◽  
Spatiotemporal Regulation ◽  
Time Lapse Imaging

AbstractThe endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.SummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

scholarly journals Cytoplasmic dynamics of myosin IIA and IIB: spatial ‘sorting’ of isoforms in locomoting cells

1998 ◽  
Vol 111 (15) ◽  
pp. 2085-2095 ◽  
Author(s):  
J. Kolega
Keyword(s):  
Cultured Cells ◽  
Myosin Ii ◽  
Cell Movement ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Immunofluorescence Staining ◽  
Spatial Sorting ◽  
Time Lapse Imaging

Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to ‘sort’ within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.

scholarly journals Monitoring ephemeral, intermittent and perennial streamflow: A data set from 182 sites in the Attert catchment, Luxembourg

2019 ◽  
Author(s):  
Nils H. Kaplan ◽  
Ernestine Sohrt ◽  
Theresa Blume ◽  
Markus Weiler
Keyword(s):  
Spatial Dynamics ◽  
Time Lapse ◽  
Time Interval ◽  
Hydrologic Models ◽  
Stream Network ◽  
Data Set ◽  
German Research ◽  
Single Dataset ◽  
Temporal And Spatial ◽  
Presence And Absence

Abstract. The temporal and spatial dynamics of streamflow presence and absence is considered vital information to many hydrological and ecological studies. Measuring the duration of active streamflow and dry periods in the channel allows us to classify the degree of intermittency of streams. We used different sensing techniques including time-lapse imagery, electric conductivity and stage measurements to generate a combined dataset of presence and absence of streamflow within various nested sub-catchments in the Attert Catchment, Luxembourg. The first sites of observation were established in 2013 and successively extended to a total number of 182 in 2016 as part of the project “Catchments As Organized Systems” (CAOS). Temporal resolution ranged from 5 to 15 minutes intervals. Each single dataset was carefully processed and quality controlled before the time interval was homogenised to 30 minutes. The dataset provides valuable information of the dynamics of a meso-scale stream network in space and time. This can be used to test and evaluate hydrologic models, but also for the assessment of the intermittent stream ecosystem in the Attert basin. The dataset presented in this paper is available at the online repository of the German Research Center for Geosciences (GFZ, https://doi.org/10.5880/FIDGEO.2019.010).

O-003 The puzzling unknowns of abnormal fertilization and first cleavage

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Racowsky
Keyword(s):  
Embryo Development ◽  
Time Lapse ◽  
Critical Event ◽  
Cleavage Division ◽  
Sequence Of Events ◽  
Polar Bodies ◽  
Abnormal Fertilization ◽  
Temporal And Spatial ◽  
Temporal Events ◽  
Time Lapse Imaging

Abstract text Fertilization is a critical event in development in that it provides the connection between the gametes and the earliest stages of embryogenesis. Yet, despite the central importance of this process in contributing to embryo developmental fate, clinical embryologists have historically assessed fertilization merely by the number of pronuclei and, if two are present, perhaps, by the presence of two polar bodies. Even though over 20 years ago, time lapse imaging was applied for defining early events of fertilization (Payne et al., 1997), it is only with contemporary time-lapse imaging systems in the last few years that detailed evaluation of spatial and temporal events of fertilization have been described (Iwata & Yasuyuki, 2016; Cottichio et al., 2018). These careful analyses allow us to describe typical and atypical events of fertilization and how they are each associated with timing of the first cleavage division and subsequent embryo development. In this lecture, we will first describe the fundamental underpinnings of fertilization and highlight the normal events associated with this process. We will then discuss gross morphological abnormalities as visualized by light microscopy and highlight the unknowns associated with these events. Finally, we will focus on time-lapse imaging studies, which have revealed the remarkable spatial and temporal coordination of meiotic resumption, pronuclear dynamics, chromatin organization and cytoplasmic/cortical modifications that occur during fertilization and the implications of aberrations for the first cleavage division. At the conclusion of this presentation, attendees should be able to: Review the normal events associated with fertilization and the first cleavage division. 1 Describe gross morphological aberrations of these two fundamental processes. 2 Discuss temporal and spatial abnormalities in the coordinated sequence of events that underly these processes. 3 State the potential application of these abnormalities as predictors of abnormal embryo development. 4 Summarize the puzzling unknowns that underly these abnormalities.

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 Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Hideaki Ando ◽  
Matsumi Hirose ◽  
Gen Kurosawa ◽  
Soren Impey ◽  
Katsuhiko Mikoshiba
Keyword(s):  
Time Lapse ◽  
Living Cells ◽  
Single Living Cells ◽  
Time Lapse Imaging ◽  
Kinetics Of ◽  
Single Living
Sign in / Sign up

Export Citation Format

Share Document