scholarly journals Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

2019 ◽  
Author(s):  
Alexandra F. Long ◽  
Pooja Suresh ◽  
Sophie Dumont
Keyword(s):  
Polymerization Rate ◽  
Dynamic Force ◽  
Force Transmission ◽  
Physical Mechanisms ◽  
Engineering Principle ◽  
Spindle Microtubules ◽  
Structural Homeostasis ◽  
Spindle Structure

AbstractAt cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus- and minus-ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

scholarly journals Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Alexandra F. Long ◽  
Pooja Suresh ◽  
Sophie Dumont
Keyword(s):  
Polymerization Rate ◽  
Dynamic Force ◽  
Force Transmission ◽  
Physical Mechanisms ◽  
Engineering Principle ◽  
Spindle Microtubules ◽  
Structural Homeostasis ◽  
Spindle Structure

At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

Factors Controlling Electropermeabilisation of Cell Membranes

2002 ◽  
Vol 1 (5) ◽  
pp. 319-327 ◽  
Author(s):  
M. P. Rols ◽  
M. Golzio ◽  
B. Gabriel ◽  
J. Teissié
Keyword(s):  
Cell Surface ◽  
Electric Field ◽  
Pulse Duration ◽  
Cell Membranes ◽  
Physical Parameters ◽  
Local Exchange ◽  
Physical Mechanisms ◽  
A Cell

Electric field pulses are a new approach for drug and gene delivery for cancer therapy. They induce a localized structural alteration of cell membranes. The associated physical mechanisms are well explained and can be safely controlled. A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. Electric field pulses with an overcritical intensity evoke a local membrane alteration. A free exchange of hydrophilic low molecular weight molecules takes place across the membrane. A leakage of cytosolic metabolites and a loading of polar drugs into the cytoplasm are obtained. The fraction of the cell surface which is competent for exchange is a function of the field intensity. The level of local exchange is strongly controlled by the pulse duration and the number of successive pulses. The permeabilised state is long lived. Its lifetime is under the control of the cumulated pulse duration. Cell viability can be preserved. Gene transfer is obtained but its mechanism is not a free diffusion. Plasmids are electrophoretically accumulated against the permeabilised cell surface and form aggregates due to the field effect. After the pulses, several steps follow: translocation to the cytoplasm, traffic to the nucleus and expression. Molecular structural and metabolic changes in cells remain mostly poorly understood. Nevertheless, while most studies were established on cells in culture ( in vitro), recent experiments show that similar effects are obtained on tissue ( in vivo). Transfer remains controlled by the physical parameters of the electrical treatment.

scholarly journals The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation

2020 ◽  
Author(s):  
Emma Carley ◽  
Rachel K. Stewart ◽  
Abigail Zieman ◽  
Iman Jalilian ◽  
Diane. E. King ◽  
...  
Keyword(s):  
Cell Fate ◽  
Control Cell ◽  
Nuclear Lamina ◽  
Epidermal Differentiation ◽  
Keratinocyte Differentiation ◽  
Linc Complex ◽  
Force Transmission ◽  
Cell Fate Decisions

AbstractWhile the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

scholarly journals An Experimental Approach to Evaluate the Dynamic Behavior of the Human Knee

1988 ◽  
Vol 110 (1) ◽  
pp. 69-73 ◽  
Author(s):  
H. W. J. Jans ◽  
L. J. M. G. Dortmans ◽  
A. A. H. J. Sauren ◽  
A. Huson
Keyword(s):  
Experimental Approach ◽  
Function Analysis ◽  
Measuring System ◽  
Dynamic Force ◽  
Force Transmission ◽  
Human Knee ◽  
Human Knee Joint ◽  
In Vitro Investigation ◽  
Transfer Function Analysis

An experimental approach for an in vitro investigation of some aspects of dynamic force transmission through the human knee joint is presented. Essentially, the behavior of the joint was analyzed by measuring the responses to low level random excitation of the tibia while the femur was clamped. A global equilibrium position of the joint was attained by exerting static forces on the tibia via three tendinous muscle attachments. The responses to the applied dynamic loads were measured using a multi-channel dynamic measuring system and quantified by means of transfer function analysis techniques. Some preliminary experimental results are presented to illustrate the effects of variation of the direction and the magnitude of the applied dynamic and static loads.

scholarly journals Implication of a novel multiprotein Dam1p complex in outer kinetochore function

2001 ◽  
Vol 155 (7) ◽  
pp. 1137-1146 ◽  
Author(s):  
Iain M. Cheeseman ◽  
Christine Brew ◽  
Michael Wolyniak ◽  
Arshad Desai ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Phosphorylation Event ◽  
Spindle Microtubules ◽  
Green Fluorescent ◽  
Kinetochore Function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

scholarly journals Cancer-associated fibroblasts actively compress cancer cells and modulate mechanotransduction

2021 ◽  
Author(s):  
Jorge Barbazan ◽  
Carlos Perez-Gonzalez ◽  
Manuel Gomez-Gonzalez ◽  
Mathieu Dedenon ◽  
Sophie Richon ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Intrinsic Property ◽  
Cancer Associated Fibroblasts ◽  
Force Transmission ◽  
Force Measurements ◽  
Cancer Cell Growth ◽  
Mechanical Signaling ◽  
Actomyosin Contractility

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule is a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.

F.05 Characterization of NBCA glue polymerization for embolization of brain AVM’s

2016 ◽  
Vol 43 (S2) ◽  
pp. S19-S20
Author(s):  
BH Wang ◽  
D Pelz ◽  
D Lee ◽  
MR Boulton ◽  
SP Lownie
Keyword(s):  
High Speed ◽  
Contact Angles ◽  
Polymerization Rate ◽  
Physical Interaction ◽  
Wide Range ◽  
Glue Injection ◽  
Glue Embolization

Background: Brain arteriovenous malformations (AVM’s) are abnormal connections between arteries and veins. Endovascular glue embolization with N-butyl cyanoacrylate (NBCA) is an accepted form of treatment, with most complications related to timing of polymerization. Current literature reports a wide range of polymerization times with large discrepancies between in-vivo and in-vitro results. Methods: Polymerization time was measured for mixtures of lipiodol/NBCA of 50/50, 60/40, 70/30. The influence of pH, temperature and presence of biological catalysts on polymerization rate was investigated in-vivo using submerged droplet tests. PVA-C, silicone and endothelium surfaces were compared and contact angles were measured to assess physical interaction with NBCA. High-speed video of glue injection through a microcatheter was captured to characterize coaxial flow. Results: Polymerization rate increases with pH and temperature. A hydrophilic substrate such as PVA-C provides surface properties that are most similar to endothelium. Endothelium provides a catalytic surface that increases the rate of polymerization. Blood products further increase the polymerization rate with RBC’s providing almost instantaneous polymerization of NBCA upon contact. Characterization of coaxial flow shows dripping to jetting transition with significant wall effect. Conclusions: We have successfully deconstructed and characterized the dynamic behavior of NBCA embolization. A refined understanding of NBCA behavior could help reduce embolization-related complications.

scholarly journals Interaction of NuMA protein with the kinesin Eg5: its possible role in bipolar spindle assembly and chromosome alignment

2013 ◽  
Vol 451 (2) ◽  
pp. 195-204 ◽  
Author(s):  
Yuko Iwakiri ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Mitotic Apparatus ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Interphase Cells ◽  
Correct Alignment ◽  
Spindle Microtubules

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro. During mitosis Eg5 appears to interact with NuMA in the vicinity of the spindle poles, whereas the interaction does not occur in interphase cells, where Eg5 is distributed throughout the cytoplasm but NuMA exclusively localizes to the nucleus. Slight, but significant, depletion of Eg5 in HeLa cells by RNA interference results in formation of less-focused spindle poles with misaligned chromosomes in metaphase; these phenotypes are similar to those induced by depletion of NuMA. Since NuMA is less accumulated at the spindle poles in Eg5-depleted cells, Eg5 probably contributes to spindle assembly via regulating NuMA localization. Furthermore, depletion of cytoplasmic dynein induces mislocalization of NuMA and phenotypes similar to those observed in NuMA-depleted cells, without affecting Eg5 localization to the spindles. Thus dynein appears to control NuMA function in conjunction with Eg5.

scholarly journals Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum.

1984 ◽  
Vol 99 (1) ◽  
pp. 155-165 ◽  
Author(s):  
T Schedl ◽  
T G Burland ◽  
K Gull ◽  
W F Dove
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Microscopic Analysis ◽  
Nucleic Acid Hybridization ◽  
Electron Microscopic ◽  
Two Dimensional Gel Electrophoresis ◽  
Spindle Microtubules ◽  
Beta 2

The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.

scholarly journals Regulation of Kif15 localization and motility by the C-terminus of TPX2 and microtubule dynamics

2017 ◽  
Vol 28 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Barbara J. Mann ◽  
Sai K. Balchand ◽  
Patricia Wadsworth
Keyword(s):  
Equatorial Region ◽  
Growth Reduction ◽  
Spindle Formation ◽  
Terminal Domain ◽  
C Terminus ◽  
Microtubule Growth ◽  
Spindle Microtubules ◽  
In Cells

Mitotic motor proteins generate force to establish and maintain spindle bipolarity, but how they are temporally and spatially regulated in vivo is unclear. Prior work demonstrated that a microtubule-associated protein, TPX2, targets kinesin-5 and kinesin-12 motors to spindle microtubules. The C-terminal domain of TPX2 contributes to the localization and motility of the kinesin-5, Eg5, but it is not known whether this domain regulates kinesin-12, Kif15. We found that the C-terminal domain of TPX2 contributes to the localization of Kif15 to spindle microtubules in cells and suppresses motor walking in vitro. Kif15 and Eg5 are partially redundant motors, and overexpressed Kif15 can drive spindle formation in the absence of Eg5 activity. Kif15-dependent bipolar spindle formation in vivo requires the C-terminal domain of TPX2. In the spindle, fluorescent puncta of GFP-Kif15 move toward the equatorial region at a rate equivalent to microtubule growth. Reduction of microtubule growth with paclitaxel suppresses GFP-Kif15 motility, demonstrating that dynamic microtubules contribute to Kif15 behavior. Our results show that the C-terminal region of TPX2 regulates Kif15 in vitro, contributes to motor localization in cells, and is required for Kif15 force generation in vivo and further reveal that dynamic microtubules contribute to Kif15 behavior in vivo.

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