scholarly journals Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

2017 ◽  
Author(s):  
César Díaz-Celis ◽  
Viviana I. Risca ◽  
Felipe Hurtado ◽  
Jessica K. Polka ◽  
Scott D. Hansen ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Complex Dynamics ◽  
Critical Concentration ◽  
Polarized Growth ◽  
Gtp Hydrolysis ◽  
Filament Dynamics ◽  
Mixed Polarity ◽  
Self Association

AbstractBacteria of the genusProsthecobacterexpress homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). ThebtubABgenes likely entered theProsthecobacterlineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterizedin vitrobMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm ofProsthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.ImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

scholarly journals Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
César Díaz-Celis ◽  
Viviana I. Risca ◽  
Felipe Hurtado ◽  
Jessica K. Polka ◽  
Scott D. Hansen ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Complex Dynamics ◽  
Critical Concentration ◽  
Polarized Growth ◽  
Gtp Hydrolysis ◽  
Content Type ◽  
Filament Dynamics ◽  
Mixed Polarity

ABSTRACT Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and β-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer. IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.

scholarly journals A method for multiprotein assembly in cells reveals independent action of kinesins in complex

2014 ◽  
Vol 207 (3) ◽  
pp. 393-406 ◽  
Author(s):  
Stephen R. Norris ◽  
Virupakshi Soppina ◽  
Aslan S. Dizaji ◽  
Kristin I. Schimert ◽  
David Sept ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Intracellular Trafficking ◽  
Intracellular Transport ◽  
Emergent Behavior ◽  
Independent Action ◽  
Dynamic Interactions ◽  
Macromolecular Complexes ◽  
Protein Components ◽  
In Cells

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track.

scholarly journals Polymerization of FtsZ, a Bacterial Homolog of Tubulin

2001 ◽  
Vol 276 (15) ◽  
pp. 11743-11753 ◽  
Author(s):  
Laura Romberg ◽  
Martha Simon ◽  
Harold P. Erickson
Keyword(s):  
Steady State ◽  
Critical Concentration ◽  
Gtp Hydrolysis ◽  
Nucleotide Hydrolysis ◽  
High Affinity ◽  
Hydrolysis Rates ◽  
Bacterial Homolog ◽  
Nucleation Phase ◽  
Maximum Steady State

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis.In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or “isodesmic”) assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolarKD.

scholarly journals Individual Molecular Motors use Low Forces to bypass Roadblocks during Collective Cargo Transport

2021 ◽  
Author(s):  
Saurabh Shukla ◽  
Alice Troitskaia ◽  
Nikhila Swarna ◽  
Barun Kumar Maity ◽  
Marco Tjioe ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Force Sensor ◽  
The Other ◽  
Cargo Transport ◽  
Different Types ◽  
Multicolor Fluorescence ◽  
High Throughput Assay

AbstractA cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesins in vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expected to be generally useful for many different types of molecular motors.

scholarly journals In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB

2005 ◽  
Vol 169 (2) ◽  
pp. 233-238 ◽  
Author(s):  
Christopher A. Sontag ◽  
James T. Staley ◽  
Harold P. Erickson
Keyword(s):  
Critical Concentration ◽  
Three Dimensional ◽  
Outer Diameter ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Assembly Mechanism ◽  
In Vitro Assembly ◽  
Cooperative Assembly ◽  
Versus Concentration

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35–36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4–7 protofilaments wide (20–30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4–1.0 μM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.

scholarly journals Self-repair protects microtubules from their destruction by molecular motors:

2018 ◽  
Author(s):  
Sarah Triclin ◽  
Daisuke Inoue ◽  
Jeremie Gaillard ◽  
Zaw Min Htet ◽  
Morgan De Santis ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Atp Hydrolysis ◽  
Low Energy ◽  
Mechanical Work ◽  
Repair Mechanism ◽  
Tubulin Dimer ◽  
Self Repair

Microtubules are dynamic polymers that are used for intracellular transport and chromosome segregation during cell division. Their instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Microtubules function in coordination with kinesin and dynein molecular motors, which use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the forces produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that the mechanical work of molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. This effect was not observed when free tubulin dimers were present in the assay. Using fluorescently labelled tubulin dimers we found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of kinesin and dynein motors and the renewal of the microtubule lattice.

scholarly journals A FLIC motility assay reveals myosin-6 coordination limited by actin filament buckling

2016 ◽  
Author(s):  
Agata K. Krenc ◽  
Jagoda J. Rokicka ◽  
Ronald S. Rock
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Motor Coordination ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Myosin Motors ◽  
Unique Application

ABSTRACTTeams of myosin motors carry out intracellular transport and contract the actin cytoskeleton. To fully understand the behavior of multi-myosin ensembles we need to know the properties of individual myosins and the mode of interaction between them. Current models of the interactions within the myosin complex treat the actin filament as a stiff rod, not contributing to the regulation of collective myosin dynamics. Here, we present data suggesting that force transduction through the actin filament is an important element of interaction within myosin-6 ensembles in vitro. Multiple myosin-6s coordinate their steps if they are separated by a short (and therefore high-force bearing) segment of actin. The measurements were performed using Fluorescence Interference Contrast Microscopy (FLIC) to measure small changes in the height of fluorescently labeled actin. Using FLIC, we assign the positions of myosins in a gliding filament assay geometry and measure their attachment time to actin. We also identify actin segments that are buckled or under tension. We show that myosin-6 holds actin about 10 nm above the surface. However, due to asynchronous myosin stepping, frequent buckles up to about 60 nm high appear. The buckle lifetime decreases as the distance between the myosin-6s is reduced, a sign of inter-motor coordination. Our data are consistent with coordinated stepping of closely spaced myosins, but uncoordinated motility with widely separated myosins where buckles can form. These features would be expected to operate on myosins in the cell, where motor spacing may vary considerably depending on the target organelle.SIGNIFICANCE STATEMENTMyosins are molecular motors that carry out intracellular transport. Interactions between the myosins are crucial for understanding their function. Using Fluorescence Interference Contrast (FLIC) microscopy we characterized the interaction between multiple myosin-6 motors immobilized to the surface of a slide and pulling the same actin filament. Our results point towards coordination of myosin steps as a mechanism governing the behavior of a multi-myosin complex. We also demonstrated the unique application of FLIC microscopy for highly parallel identification and measurement of single myosin motors in a gliding filament format. These features of FLIC enable a robust study of collective myosin dynamics.

scholarly journals Modelling cytoskeletal transport by clusters of non-processive molecular motors with limited binding sites

2020 ◽  
Vol 7 (8) ◽  
pp. 200527
Author(s):  
Naruemon Rueangkham ◽  
Ian D. Estabrook ◽  
Rhoda J. Hawkins
Keyword(s):  
Molecular Motors ◽  
Binding Sites ◽  
Intracellular Transport ◽  
Exclusion Process ◽  
Stochastic Simulations ◽  
Published Data ◽  
Analytical Work ◽  
Number Of Binding Sites

Molecular motors are responsible for intracellular transport of a variety of biological cargo. We consider the collective behaviour of a finite number of motors attached on a cargo. We extend previous analytical work on processive motors to the case of non-processive motors, which stochastically bind on and off cytoskeletal filaments with a limited number of binding sites available. Physically, motors attached to a cargo cannot bind anywhere along the filaments, so the number of accessible binding sites on the filament should be limited. Thus, we analytically study the distribution and the velocity of a cluster of non-processive motors with limited number of binding sites. To validate our analytical results and to go beyond the level of detail possible analytically, we perform Monte Carlo latticed based stochastic simulations. In particular, in our simulations, we include sequence preservation of motors performing stepping and binding obeying a simple exclusion process. We find that limiting the number of binding sites reduces the probability of non-processive motors binding but has a relatively small effect on force–velocity relations. Our analytical and stochastic simulation results compare well to published data from in vitro and in vivo experiments.

scholarly journals In vitro reconstitution reveals phosphoinositides as cargo-release factors and activators of the ARF6 GAP ADAP1

2020 ◽  
Vol 118 (1) ◽  
pp. e2010054118
Author(s):  
Christian Duellberg ◽  
Albert Auer ◽  
Nikola Canigova ◽  
Katrin Loibl ◽  
Martin Loose
Keyword(s):  
Molecular Motors ◽  
Small Gtpase ◽  
Complex Dynamic ◽  
Gtp Hydrolysis ◽  
Dendrite Branching ◽  
Precise Control ◽  
In Vitro Reconstitution ◽  
Adp Ribosylation Factor ◽  
Dynamic Interplay

The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1’s functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3as well as PI(3,4)P2act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1’s enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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