scholarly journals Measuring Force Generation within Reconstituted Microtubule Bundle Assemblies using Optical Tweezers

Cytoskeleton ◽  
2021 ◽  
Author(s):  
Omayma Al Azzam ◽  
Cameron Lee Trussell ◽  
Dana N. Reinemann
Keyword(s):  
Optical Tweezers ◽  
Force Generation ◽  
Microtubule Bundle ◽  
Measuring Force

FORCE GENERATION BY MICROTUBULE BUNDLES

2009 ◽  
Vol 04 (01n02) ◽  
pp. 33-43
Author(s):  
JULIEN HUSSON ◽  
LIEDEWIJ LAAN ◽  
MARILEEN DOGTEROM
Keyword(s):  
Polyethylene Glycol ◽  
Optical Tweezers ◽  
Force Generation ◽  
Mitotic Chromosomes ◽  
Microtubule Bundle ◽  
Microtubule Associated Proteins ◽  
Complex Processes ◽  
Associated Proteins ◽  
Dynamic Microtubule

Dynamic microtubule bundles are involved in several motility processes in cells, for instance by interacting with mitotic chromosomes. The study of microtubule bundle dynamics and force generation can help understanding the mechanisms underlying these complex processes. Here we discuss experimental results on the force-generating capabilities of bundles of non-crosslinked growing microtubules obtained using an optical tweezers technique in vitro. We discuss the possible effects on force generation by these microtubule bundles of specific (with microtubule-associated proteins) and aspecific (with ions or crowding agents) ways of crosslinking microtubules. We present preliminary results showing that force generation by microtubule bundles is enhanced in the presence of polyethylene glycol used as a crowding agent, and discuss possible explanations for this observation.

scholarly journals Detailed analyses of stall force generation inMycoplasma mobilegliding

2017 ◽  
Author(s):  
Masaki Mizutani ◽  
Isil Tulum ◽  
Yoshiaki Kinosita ◽  
Takayuki Nishizaka ◽  
Makoto Miyata
Keyword(s):  
Optical Tweezers ◽  
Type Strain ◽  
Wild Type Strain ◽  
Gear Ratio ◽  
Solid Surfaces ◽  
Force Generation ◽  
Wild Type ◽  
Step Size ◽  
Large Gear ◽  
Stall Force

ABSTRACTMycoplasma mobileis a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 µm/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP; the units catch and pull on sialylated oligosaccharides fixed to solid surfaces. In the present study, we measured the stall force of wild-type and mutant strains ofM. mobilecarrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 pN following the addition of 0–0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. Following the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 pN. Considering the 70-nm step size, this small unit force may be explained by the large gear ratio involved in theM. mobilegliding machinery.SIGNIFICANCEMycoplasmais a genus of bacteria that parasitizes animals. Dozens ofMycoplasmaspecies glide over the tissues of their hosts during infection. The gliding machinery ofMycoplasma mobile, the fastest species, includes intracellular motors and hundreds of legs on the cell surface. In the present study, we precisely measured force generation using a highly focused laser beam arrangement (referred to as optical tweezers) under various conditions. The measurements obtained in this study suggest that the rapid gliding exhibited byM. mobilearises from the large gear ratio of its gliding machinery.

scholarly journals Pathogenic mutations in the kinesin-3 motor KIF1A diminish force generation and movement through allosteric mechanisms

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Breane G. Budaitis ◽  
Shashank Jariwala ◽  
Lu Rao ◽  
Yang Yue ◽  
David Sept ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Neurodevelopmental Disorders ◽  
Force Generation ◽  
Long Distance ◽  
Long Distance Transport ◽  
Cargo Transport ◽  
Dynamics Simulations ◽  
Run Lengths ◽  
Allosteric Mechanisms ◽  
Distance Transport

The kinesin-3 motor KIF1A functions in neurons, where its fast and superprocessive motility facilitates long-distance transport, but little is known about its force-generating properties. Using optical tweezers, we demonstrate that KIF1A stalls at an opposing load of ~3 pN but more frequently detaches at lower forces. KIF1A rapidly reattaches to the microtubule to resume motion due to its class-specific K-loop, resulting in a unique clustering of force generation events. To test the importance of neck linker docking in KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders. Molecular dynamics simulations predict that V8M and Y89D mutations impair neck linker docking. Indeed, both mutations dramatically reduce the force generation of KIF1A but not the motor’s ability to rapidly reattach to the microtubule. Although both mutations relieve autoinhibition of the full-length motor, the mutant motors display decreased velocities, run lengths, and landing rates and delayed cargo transport in cells. These results advance our understanding of how mutations in KIF1A can manifest in disease.

scholarly journals Force production of human cytoplasmic dynein is limited by its processivity

2020 ◽  
Vol 6 (15) ◽  
pp. eaaz4295 ◽  
Author(s):  
Sibylle Brenner ◽  
Florian Berger ◽  
Lu Rao ◽  
Matthew P. Nicholas ◽  
Arne Gennerich
Keyword(s):  
Optical Tweezers ◽  
Molecular Motor ◽  
Force Production ◽  
Cytoplasmic Dynein ◽  
Force Generation ◽  
New Paradigm ◽  
Average Force ◽  
Complex Motor ◽  
Hyperbolic Curve ◽  
Stall Force

Cytoplasmic dynein is a highly complex motor protein that generates forces toward the minus end of microtubules. Using optical tweezers, we demonstrate that the low processivity (ability to take multiple steps before dissociating) of human dynein limits its force generation due to premature microtubule dissociation. Using a high trap stiffness whereby the motor achieves greater force per step, we reveal that the motor’s true maximal force (“stall force”) is ~2 pN. Furthermore, an average force versus trap stiffness plot yields a hyperbolic curve that plateaus at the stall force. We derive an analytical equation that accurately describes this curve, predicting both stall force and zero-load processivity. This theoretical model describes the behavior of a kinesin motor under low-processivity conditions. Our work clarifies the true stall force and processivity of human dynein and provides a new paradigm for understanding and analyzing molecular motor force generation for weakly processive motors.

scholarly journals Pathogenic Mutations in the Kinesin-3 Motor KIF1A Diminish Force Generation and Movement Through Allosteric Mechanisms

2020 ◽  
Author(s):  
Breane G. Budaitis ◽  
Shashank Jariwala ◽  
Lu Rao ◽  
David Sept ◽  
Kristen J. Verhey ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Motor Domain ◽  
Force Generation ◽  
Long Distance ◽  
C Elegans ◽  
Long Distance Transport ◽  
Dynamics Simulations ◽  
Run Lengths ◽  
Allosteric Mechanisms ◽  
Kinesin Superfamily

ABSTRACTThe kinesin-3 motor KIF1A functions in neurons where its fast and superprocessive motility is thought to be critical for long-distance transport. However, little is known about the force-generating properties of kinesin-3 motors. Using optical tweezers, we demonstrate that KIF1A and its C. elegans homolog UNC-104 undergo force-dependent detachments at ~3 pN and then rapidly reattach to the microtubule to resume motion, resulting in a sawtooth pattern of clustered force generation events that is unique among the kinesin superfamily. Whereas UNC-104 motors stall before detaching, KIF1A motors do not. To examine the mechanism of KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders, V8M and Y89D, based on their location in structural elements required for force generation in kinesin-1. Molecular dynamics simulations predict that the V8M and Y89D mutations impair docking of the N-terminal (β9) or C-terminal (β10) portions of the neck linker, respectively, to the KIF1A motor domain. Indeed, both mutations dramatically impair force generation of KIF1A but not the motor’s ability to rapidly reattach to the microtubule track. Homodimeric and heterodimeric mutant motors also display decreased velocities, run lengths, and landing rates and homodimeric Y89D motors exhibit a higher frequency of non-productive, diffusive events along the microtubule. In cells, cargo transport by the mutant motors is delayed. Our work demonstrates the importance of the neck linker in the force generation of kinesin-3 motors and advances our understanding of how mutations in the kinesin motor domain can manifest in disease.

‘Corkscrewing’, as evidence for force generation within a detergent-extracted microtubule translocation system from insect ovaries

1989 ◽  
Vol 92 (1) ◽  
pp. 21-27
Author(s):  
H. Stebbings ◽  
K.K. Sharma
Keyword(s):  
Electrophoretic Analysis ◽  
Force Generation ◽  
Microtubule Bundle ◽  
Microtubule Associated Proteins ◽  
Microtubule Motor ◽  
Associated Proteins ◽  
Hemipteran Insect ◽  
Helical Configuration

Microtubule-based translocation channels within the ovaries of the hemipteran insect Notonecta have been isolated by microdissection, and then detergent-extracted to leave a bundle of some 30,000 aligned microtubules. On addition of ATP and other hydrolysable nucleotides the microtubule bundle contorts into a helical configuration, a property we have called ‘corkscrewing’, before straightening again. This we believe to be indicative of force generation within the bundle. Electrophoretic analysis of the bundle of native microtubules reveals many polypeptides apart from the tubulins, and a number of these comigrate with microtubule-associated proteins (MAPs), which copolymerize with tubulins in reassembled microtubules from the same system. Corkscrewing does not occur if the microtubule bundle is pretreated with salt, a procedure that removes MAPs from microtubules, suggesting that the force is generated by a MAP or MAPs. In addition, certain minor polypeptides comprising the native microtubules are ATP-sensitive, a property expected of a microtubule motor.

scholarly journals Synergy between RecBCD subunits is essential for efficient DNA unwinding

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rani Zananiri ◽  
Omri Malik ◽  
Sergei Rudnizky ◽  
Vera Gaydar ◽  
Roman Kreiserman ◽  
...  
Keyword(s):  
Dna Binding ◽  
Optical Tweezers ◽  
Binding Proteins ◽  
Strand Break ◽  
Dna Binding Proteins ◽  
The Other ◽  
Force Generation ◽  
Atp Binding ◽  
Dna Unwinding

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD’s individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25–40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.

scholarly journals Viscoelastic dissipation stabilizes cell shape changes during tissue morphogenesis

2017 ◽  
Author(s):  
R Clément ◽  
C. Collinet ◽  
B. Dehapiot ◽  
T. Lecuit ◽  
P.-F. Lenne
Keyword(s):  
Optical Tweezers ◽  
Cell Shape ◽  
Myosin Ii ◽  
Force Generation ◽  
Tissue Morphogenesis ◽  
Cell Shape Changes ◽  
Cellular Forces ◽  
Transient Forces ◽  
Shape Changes

Tissue morphogenesis relies on the production of active cellular forces. Understanding how such forces are mechanically converted into cell shape changes is essential to our understanding of morphogenesis. Here we use Myosin II pulsatile activity during Drosophila embryogenesis to study how transient forces generate irreversible cell shape changes. Analyzing the dynamics of junction shortening and elongation resulting from Myosin II pulses, we find that long pulses yield less reversible deformations, typically a signature of dissipative mechanics. This is consistent with a simple viscoelastic description, which we use to model individual shortening and elongation events. The model predicts that dissipation typically occurs on the minute timescale, a timescale commensurate with that of force generation by Myosin II pulses. We test this estimate by applying time-controlled forces on junctions with optical tweezers. Our results argue that active junctional deformation is stabilized by dissipation. Hence, tissue morphogenesis requires coordination between force generation and dissipation.

Defining the trapping limits of holographical optical tweezers

2004 ◽  
Vol 51 (3) ◽  
pp. 409-414 ◽  
Author(s):  
P. Jordan ◽  
J. Leach ◽  
M. J. Padgett ◽  
J. Cooper ◽  
G. Sinclair
Keyword(s):  
Optical Tweezers
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