scholarly journals Roles of motor on-rate and cargo mobility in intracellular transport

2020 ◽  
Author(s):  
Matthew J. Bovyn ◽  
Babu J.N. Reddy ◽  
Steven P. Gross ◽  
Jun F. Allard
Keyword(s):  
Modeling And Simulation ◽  
Single Molecule ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Microtubule Associated Proteins ◽  
Single Motor ◽  
Two Factors ◽  
Associated Proteins ◽  
Binding Time ◽  
The Difference

AbstractMolecular motors like kinesin are critical for cellular organization and biological function including in neurons. There is detailed understanding of how they move and how factors such as applied force and the presence of microtubule-associated proteins can alter this single-motor travel. In order to walk, the cargo-motor complex must first attach to a microtubule. This attachment process is less studied. Here, we use a combination of single-molecule bead experiments, modeling, and simulation to examine how cargos with kinesin-1 bind to microtubules. In experiment, we find that increasing cargo size and environment viscosity both signficantly slow cargo binding time. We use modeling and simulation to examine how the single motor on rate translates to the on rate of the cargo. Combining experiment and modeling allows us to estimate the single motor on rate as 100 s−1. This is a much higher value than previous estimates. We attribute the difference between our measurements and previous estimates to two factors: first, we are directly measuring initial motor attachment (as opposed to re-binding of a second motor) and second, the theoretical framework allows us to account for missed events (i.e. binding events not detected by the experiments due to their short duration). This indicates that the mobility of the cargo itself, determined by its size and interaction with the cytoplasmic environment, play a previously underestimated role in determining intracellular transport kinetics.

scholarly journals The mechanism of motor inhibition by microtubule-associated proteins

2020 ◽  
Author(s):  
Luke S Ferro ◽  
Lisa Eshun-Wilson ◽  
Mert Gölcük ◽  
Jonathan Fernandes ◽  
Teun Huijben ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Intracellular Transport ◽  
List Type ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Projection Domain ◽  
Electron Imaging

SUMMARYMicrotubule (MT)-associated proteins (MAPs) regulate intracellular transport by selectively recruiting or excluding kinesin and dynein motors from MTs. We used single-molecule and cryo-electron imaging to determine the mechanism of MAP-motor interactions in vitro. Unexpectedly, we found that the regulatory role of a MAP cannot be predicted based on whether it overlaps with the motor binding site or forms liquid condensates on the MT. Although the MT binding domain (MTBD) of MAP7 overlaps with the kinesin-1 binding site, tethering of kinesin-1 by the MAP7 projection domain supersedes this inhibition and results in biphasic regulation of kinesin-1 motility. Conversely, the MTBD of tau inhibits dynein motility without overlapping with the dynein binding site or by forming tau islands on the MT. Our results indicate that MAPs sort intracellular cargos moving in both directions, as neither dynein nor kinesin can walk on a MAP-coated MT without favorably interacting with that MAP.HIGHLIGHTSMAP7 binds to a novel site and can coexist with tau on the MT.Kinesin-1 motility is biphasically regulated by MAP7 accumulation on the microtubule.MT decoration of MAPs inhibits motors even when they do not block the motor binding site.Motors need to interact with a MAP to walk on MAP-decorated MTs

scholarly journals Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1834
Author(s):  
Tomoko Okada ◽  
Toshihiko Ogura
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Related Gene ◽  
Autophagosome Formation ◽  
Intact Cells ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Difference ◽  
Related Proteins ◽  
Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

scholarly journals KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle

2019 ◽  
Vol 2 (1) ◽  
pp. e201800169 ◽  
Author(s):  
Heidi LH Malaby ◽  
Dominique V Lessard ◽  
Christopher L Berger ◽  
Jason Stumpff
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Binding Proteins ◽  
Mitotic Chromosome ◽  
Chromosome Movement ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Chromosome Alignment ◽  
Associated Proteins ◽  
Fiber Dynamics

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber–binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

Single-molecule mechanical study of an autonomous artificial translational molecular motor beyond bridge-burning design

Nanoscale ◽  
2021 ◽  
Author(s):  
Xinpeng Hu ◽  
Xiaodan Zhao ◽  
Iong Ying Loh ◽  
Jie Yan ◽  
Zhisong Wang
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
Molecular Motor ◽  
Force Generation ◽  
Single Motor ◽  
Mechanical Study

A key capability of molecular motors is sustainable force generation by a single motor copy. Direct force characterization at single-motor level is still missing for artificial molecular motors, though long...

Microtubule associated proteins in microtubule preparations made with and without glycerol

1984 ◽  
Vol 62 (9) ◽  
pp. 803-813 ◽  
Author(s):  
Robert A. B. Keates
Keyword(s):  
Binding Assay ◽  
Critical Concentration ◽  
Brain Homogenate ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
The Difference

Preparation of microtubule protein in the presence or absence of glycerol results in differences in polymerization properties and content of microtubule associated proteins. The variation in properties appears to result from the reduced proportion of microtubule associated proteins in preparations made with glycerol. I have used the colchicine binding assay to monitor recovery of active tubulin and have found that a single factor can account for the difference. During the in vitro assembly of microtubules from the crude brain homogenate, glycerol promotes polymerization of the bulk of the tubulin, while less than half is incorporated into microtubules in the absence of glycerol. Assembly of partly purified microtubule protein is not enhanced by glycerol however. Microtubule associated proteins present in the crude homogenate are almost completely incorporated into the microtubules regardless of the presence of glycerol, and their high content in glycerol-free preparations appears to be the trivial result of low tubulin recovery. The high affinity of microtubule associated proteins for the assembled microtubules has other consequences for in vitro studies of microtubule assembly, and critical concentration plots to determine the polymerization equilibrium constant can be distorted unless the preparation used has a high content of microtubule associated proteins.

scholarly journals Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

2021 ◽  
Author(s):  
Joyce C.M. Meiring ◽  
Ilya Grigoriev ◽  
Wilco Nijenhuis ◽  
Lukas C. Kapitein ◽  
Anna Akhmanova
Keyword(s):  
Intracellular Transport ◽  
Experimental Manipulation ◽  
Microtubule Depolymerization ◽  
Microtubule Associated Proteins ◽  
Microtubule Severing ◽  
Associated Proteins ◽  
Dividing Cells ◽  
Chromosome Separation ◽  
Organelle Morphology ◽  
Spatiotemporal Control

Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

scholarly journals Direct imaging of intraflagellar-transport turnarounds reveals that motors detach, diffuse, and reattach to opposite-direction trains

2021 ◽  
Vol 118 (45) ◽  
pp. e2115089118
Author(s):  
Zhiqing Zhang ◽  
Noémie Danné ◽  
Bonno Meddens ◽  
Iddo Heller ◽  
Erwin J. G. Peterman
Keyword(s):  
Single Molecule ◽  
Opposite Direction ◽  
Intracellular Transport ◽  
Imaging System ◽  
Intraflagellar Transport ◽  
Direct Consequence ◽  
Underlying Mechanism ◽  
Single Motor ◽  
Single Molecule Analysis ◽  
And Diffusion

Intraflagellar transport (IFT), a bidirectional intracellular transport mechanism in cilia, relies on the cooperation of kinesin-2 and IFT-dynein motors. In Caenorhabditis elegans chemosensory cilia, motors undergo rapid turnarounds to effectively work together in driving IFT. Here, we push the envelope of fluorescence imaging to obtain insight into the underlying mechanism of motor turnarounds. We developed an alternating dual-color imaging system that allows simultaneous single-molecule imaging of kinesin-II turnarounds and ensemble imaging of IFT trains. This approach allowed direct visualization of motor detachment and reattachment during turnarounds and accordingly demonstrated that the turnarounds are actually single-motor switching between opposite-direction IFT trains rather than the behaviors of motors moving independently of IFT trains. We further improved the time resolution of single-motor imaging up to 30 ms to zoom into motor turnarounds, revealing diffusion during motor turnarounds, which unveils the mechanism of motor switching trains: detach–diffuse–attach. The subsequent single-molecule analysis of turnarounds unveiled location-dependent diffusion coefficients and diffusion times for both kinesin-2 and IFT-dynein motors. From correlating the diffusion times with IFT train frequencies, we estimated that kinesins tend to attach to the next train passing in the opposite direction. IFT-dynein, however, diffuses longer and lets one or two trains pass before attaching. This might be a direct consequence of the lower diffusion coefficient of the larger IFT-dynein. Our results provide important insights into how motors can cooperate to drive intracellular transport.

scholarly journals MAP7 recruits kinesin-1 to microtubules to direct organelle transport

2019 ◽  
Author(s):  
Abdullah R. Chaudhary ◽  
Hailong Lu ◽  
Elena B. Krementsova ◽  
Carol S. Bookwalter ◽  
Kathleen M. Trybus ◽  
...  
Keyword(s):  
Single Molecule ◽  
Organelle Transport ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Binding Rate ◽  
Associated Proteins ◽  
Bidirectional Transport ◽  
Directed Motility ◽  
Clear Shift

Microtubule-associated proteins (MAPs) play well-characterized roles in regulating microtubule polymerization, dynamics, and organization. In addition, MAPs control trans-port along microtubules by regulating the motility of kinesin and dynein. MAP7 (ensconsin, E-MAP-115) is a ubiquitous MAP that organizes the microtubule cytoskeleton in mitosis and neuronal branching. MAP7 also promotes the interaction of kinesin-1 with microtubules. We expressed and purified full-length kinesin-1 and MAP7 in Sf9 cells. In single-molecule motiity assays, MAP7 recruits kinesin-1 to microtubules, increasing the frequency of both diffusive and processive runs. Optical trapping assays on beads transported by single and teams of kinesin-1 motors indicate that MAP7 increases the relative binding rate of kinesin-1 and the number of motors simultaneously engaged in ensembles. To examine the role of MAP7 in regulating bidirectional transport, we isolated late phagosomes along with their native set of kinesin-1, kinesin-2, and dynein motors. Bidirectional cargoes exhibit a clear shift towards plus-end directed motility on MAP7-decorated microtubules due to increased forces exerted by kinesin teams. Collectively, our results indicate that MAP7 enhances kinesin-1 recruitment to microtubules and targets organelle transport to the plus end.

scholarly journals Reconstitution of dynein transport to the microtubule plus end by kinesin

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Anthony J Roberts ◽  
Brian S Goodman ◽  
Samara L Reck-Peterson
Keyword(s):  
Single Molecule ◽  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Directed Motion ◽  
Microtubule Associated Proteins ◽  
Spatial Regulation ◽  
Intracellular Movement ◽  
Associated Proteins ◽  
Directed Motility ◽  
Dynamic Microtubule

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track.

Tubulin polyglutamylase: isozymic variants and regulation during the cell cycle in HeLa cells

1999 ◽  
Vol 112 (23) ◽  
pp. 4281-4289 ◽  
Author(s):  
C. Regnard ◽  
E. Desbruyeres ◽  
P. Denoulet ◽  
B. Edde
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Molecular Motors ◽  
Dependent Manner ◽  
Proliferating Cells ◽  
Beta Tubulin ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
A Cell ◽  
Cycle Dependent

Polyglutamylation is a posttranslational modification of tubulin that is very common in neurons and ciliated or flagellated cells. It was proposed to regulate the binding of microtubule associated proteins (MAPs) and molecular motors as a function of the length of the polyglutamyl side-chain. Though much less common, this modification of tubulin also occurs in proliferating cells like HeLa cells where it is associated with centrioles and with the mitotic spindle. Recently, we partially purified tubulin polyglutamylase from mouse brain and described its enzymatic properties. In this work, we focused on tubulin polyglutamylase activity from HeLa cells. Our results support the existence of a tubulin polyglutamylase family composed of several isozymic variants specific for alpha- or beta-tubulin subunits. In the latter case, the specificity probably also concerns the different beta-tubulin isotypes. Interestingly, we found that tubulin polyglutamylase activity is regulated in a cell cycle dependent manner and peaks in G(2)-phase while the level of glutamylated tubulin peaks in mitosis. Consistent results were obtained by treating the cells with hydroxyurea, nocodazole or taxotere. In particular, in mitotic cells, tubulin polyglutamylase activity was always low while glutamylation level was high. Finally, tubulin polyglutamylase activity and the level of glutamylated tubulin appeared to be inversely related. This paradox suggests a complex regulation of both tubulin polyglutamylase and the reverse deglutamylase activity.

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