scholarly journals Spherical network contraction forms microtubule asters in confinement

Soft Matter ◽  
2018 ◽  
Vol 14 (6) ◽  
pp. 901-909 ◽  
Author(s):  
Michael P. N. Juniper ◽  
Marian Weiss ◽  
Ilia Platzman ◽  
Joachim P. Spatz ◽  
Thomas Surrey
Keyword(s):  
Motor Proteins ◽  
Living Cells ◽  
Filament Networks

Microtubules and motor proteins form active filament networks that are critical for a variety of functions in living cells.

scholarly journals Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell

2015 ◽  
Vol 112 (18) ◽  
pp. 5720-5725 ◽  
Author(s):  
Srujana Neelam ◽  
T. J. Chancellor ◽  
Yuan Li ◽  
Jeffrey A. Nickerson ◽  
Kyle J. Roux ◽  
...  
Keyword(s):  
Motor Activity ◽  
Molecular Motor ◽  
Living Cells ◽  
Elastic Response ◽  
Nuclear Surface ◽  
Mechanical Forces ◽  
Domain Protein ◽  
Mechanical Homeostasis ◽  
Filament Networks ◽  
Pulling Force

How cells maintain nuclear shape and position against various intracellular and extracellular forces is not well understood, although defects in nuclear mechanical homeostasis are associated with a variety of human diseases. We estimated the force required to displace and deform the nucleus in adherent living cells with a technique to locally pull the nuclear surface. A minimum pulling force of a few nanonewtons—far greater than typical intracellular motor forces—was required to significantly displace and deform the nucleus. Upon force removal, the original shape and position were restored quickly within a few seconds. This stiff, elastic response required the presence of vimentin, lamin A/C, and SUN (Sad1p, UNC-84)-domain protein linkages, but not F-actin or microtubules. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we conclude that the intermediate filament networks maintain nuclear mechanical homeostasis against localized forces.

scholarly journals Extreme value analysis of intracellular cargo transport by motor proteins

2021 ◽  
Author(s):  
Takuma Naoi ◽  
Yuki Kagawa ◽  
Kimiko Nagino ◽  
Shinsuke Niwa ◽  
Kumiko Hayashi
Keyword(s):  
Cell Body ◽  
Motor Proteins ◽  
Living Cells ◽  
Extreme Value ◽  
Extreme Value Analysis ◽  
Disaster Prevention ◽  
Value Analysis ◽  
Axon Terminals ◽  
Cargo Transport ◽  
Synaptic Region

In the long axon of a neuron, cargo transport between the cell body and terminal synaptic region are mainly supported by the motor proteins kinesin and dynein, which are nano-sized drivers. Synaptic materials packed as cargos are anterogradely transported to the synaptic region by kinesin, whereas materials accumulated at the axon terminals are returned to the cell body by dynein. Extreme value analysis, typically used for disaster prevention in our society, was applied to analyze the velocity of kinesin and dynein nanosized drivers to disclose their physical properties in living cells.

Nanomechanics = biomechanics

2009 ◽  
Vol 57 (1) ◽  
pp. 47-53
Author(s):  
L. Skubiszak
Keyword(s):  
Actin Filament ◽  
Conformational Changes ◽  
Energy Production ◽  
Motor Proteins ◽  
Mechanical Energy ◽  
Living Cells ◽  
Prevailing View

Nanomechanics = biomechanicsThe knowledge of the mechanism of mechanical energy production by the so-called bioengines, living cells, could be very helpful for resolving different tasks concerning nanomechanics, e.g., construction of nanorobots. The present work considers a new idea, namely that the conformational changes within the so-called track, actin filament or microtubule are crucial for production of the mechanical energy by all bioengines. This concept contrasts with the presently prevailing view, according to which the force is generated as a result of conformational changes within the so-called motor proteins: myosin, kinesin or dynein.

scholarly journals Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

1998 ◽  
Vol 143 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Miri Yoon ◽  
Robert D. Moir ◽  
Veena Prahlad ◽  
Robert D. Goldman
Keyword(s):  
Intermediate Filament ◽  
Cell Shape ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
A Cell ◽  
Filament Networks ◽  
Green Fluorescent ◽  
Vimentin Intermediate Filament

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

scholarly journals Stepwise Movements in Vesicle Transport of HER2 by Motor Proteins in Living Cells

2007 ◽  
Vol 92 (11) ◽  
pp. 4109-4120 ◽  
Author(s):  
Tomonobu M. Watanabe ◽  
Hideo Higuchi
Keyword(s):  
Motor Proteins ◽  
Living Cells ◽  
Vesicle Transport

Detection of cytokeratin dynamics by time-lapse fluorescence microscopy in living cells

1999 ◽  
Vol 112 (24) ◽  
pp. 4521-4534 ◽  
Author(s):  
R. Windoffer ◽  
R.E. Leube
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Cell Periphery ◽  
Filament Bundles ◽  
Cytokeratin 13 ◽  
Filament Networks ◽  
Green Fluorescent

To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells. Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin 13 and is therefore a reliable in vivo tag for this polypeptide and the structures formed by it. Time-lapse fluorescence microscopy reveals that the cytokeratin 13-containing network is in constant motion, resulting in continuous restructuring occurring in single and migratory cells, as well as in desmosome-anchored cells. Two major types of movement are distinguished: (i) oscillations of mostly long filaments, and (ii) an inward-directed flow of fluorescence originating as diffuse material at the cell periphery and moving in the form of dots and thin filaments toward the deeper cytoplasm where it coalesces with other filaments and filament bundles. Both movements are energy dependent and can be inhibited by nocodazole, but not by cytochalasin D. Finally, disassembly and reformation of cytokeratin filament networks are documented in dividing cells revealing distinct and rapidly occurring stages of cytokeratin organisation and distribution.

scholarly journals The SPIRE1 actin nucleator coordinates actin/myosin functions in the regulation of mitochondrial motility

2020 ◽  
Author(s):  
Felix Straub ◽  
Tobias Welz ◽  
Hannah Alberico ◽  
Rafael Oliveira Brandão ◽  
Anna Huber ◽  
...  
Keyword(s):  
Actin Filament ◽  
Motor Proteins ◽  
Transport Processes ◽  
Subcellular Localisation ◽  
Protein Targets ◽  
Energy Demands ◽  
Mitochondrial Motility ◽  
Transient Overexpression ◽  
Filament Networks ◽  
Actin Nucleator

AbstractSubcellular localisation of mitochondria provides a spatial and temporal organisation for cellular energy demands. Long-range mitochondrial transport is mediated by microtubule tracks and associated dynein and kinesin motor proteins. The actin cytoskeleton has a more versatile role and provides transport, tethering, and anchoring functions. SPIRE actin nucleators organise actin filament networks at vesicle membranes, which serve as tracks for myosin 5 motor protein-driven transport processes. Following alternative splicing, SPIRE1 is targeted to mitochondria. In analogy to vesicular SPIRE functions, we have analysed whether SPIRE1 regulates mitochondrial motility. By tracking mitochondria of living fibroblast cells from SPIRE1 mutant mice and splice-variant specific mitochondrial SPIRE1 knockout mice, we determined that the loss of SPIRE1 function increased mitochondrial motility. The SPIRE1 mutant phenotype was reversed by transient overexpression of mitochondrial SPIRE1, which almost completely inhibited motility. Conserved myosin 5 and formin interaction motifs contributed to this inhibition. Consistently, mitochondrial SPIRE1 targeted myosin 5 motors and formin actin filament generators to mitochondria. Our results indicate that SPIRE1 organises an actin/myosin network at mitochondria, which opposes mitochondrial motility.Summary statementThe mitochondrial SPIRE1 protein targets myosin 5 motor proteins and formin actin-filament nucleators/elongators towards mitochondria and negatively regulates mitochondrial motility.

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

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