Expression of a minus-end-directed motor protein induces Sf9 cells to form axon-like processes with uniform microtubule polarity orientation

1997 ◽  
Vol 110 (19) ◽  
pp. 2373-2380
Author(s):  
D.J. Sharp ◽  
R. Kuriyama ◽  
R. Essner ◽  
P.W. Baas
Keyword(s):  
Molecular Motors ◽  
Mitotic Spindle ◽  
Morphological Characteristics ◽  
Motor Protein ◽  
Motor Domain ◽  
Sf9 Cells ◽  
Atp Binding Site ◽  
Microtubule Polarity ◽  
Process Formation ◽  
Induce Process

Neurons extend two types of processes with distinct morphologies and patterns of microtubule polarity orientation. Axons are thin cylindrical processes containing microtubules that are uniformly oriented with their plus-ends-distal to the cell body while dendrites are stout tapering processes that contain nonuniformly oriented microtubules. We have proposed that these distinct microtubule patterns are established by molecular motors that transport microtubules into each type of process with the appropriate orientation. To test the feasibility of this proposal, we have embarked on a series of studies involving the expression of vertebrate motors in insect Sf9 cells. We previously focused on a kinesin-related protein termed CHO1/MKLP1, which localizes to the midzone of the mitotic spindle, and which has been shown to have the appropriate properties to transport microtubules of opposite orientation relative to one another. Expression of a fragment of CHO1/MKLP1 containing its motor domain induces Sf9 cells to extend processes with a stout tapering morphology and a nonuniform microtubule polarity pattern similar to dendrites. Here we focus on a minus-end-directed kinesin-related motor protein termed CHO2, which localizes to the non-overlapping regions of the mitotic spindle, and which has been shown to have the appropriate properties to transport microtubules with plus-ends-leading. Sf9 cells induced to express a fragment of CHO2 containing its motor domain extend processes with a long cylindrical morphology and a uniformly plus-end-distal microtubule polarity pattern similar to axons. These results show that motor proteins have the capacity to organize distinct patterns of microtubule polarity orientation during process outgrowth, and that these patterns are intimately related to the unique morphological characteristics of the processes. Moreover, mutation of three amino acids corresponding to the ATP binding site necessary for motor function suppresses the capacity of the CHO2 fragment to induce process formation and microtubule reorganization, indicating that at least in the case of CHO2, the transport properties of the motor are essential for it to elicit these effects.

scholarly journals A gelation transition enables the self-organization of bipolar metaphase spindles

2021 ◽  
Author(s):  
Benjamin A. Dalton ◽  
David Oriola ◽  
Franziska Decker ◽  
Frank Jülicher ◽  
Jan Brugués
Keyword(s):  
Molecular Motors ◽  
Mitotic Spindle ◽  
Large Scale ◽  
Relaxation Times ◽  
Network Connectivity ◽  
The Self ◽  
Self Organization ◽  
Bipolar Structure ◽  
Microtubule Polarity ◽  
Microtubule Transport

The mitotic spindle is a highly dynamic bipolar structure that emerges from the self-organization of microtubules, molecular motors, and other proteins. Sustained motor-driven poleward flows of short dynamic microtubules play a key role in the bipolar organization of spindles. However, it is not understood how the local activity of motor proteins generates these large-scale coherent poleward flows. Here, we combine experiments and simulations to show that a gelation transition enables long-ranged microtubule transport causing spindles to self-organize into two oppositely polarized microtubule gels. Laser ablation experiments reveal that local active stresses generated at the spindle midplane propagate through the structure thereby driving global coherent microtubule flows. Simulations show that microtubule gels undergoing rapid turnover can exhibit long stress relaxation times, in agreement with the long-ranged flows observed in experiments. Finally, we show that either disrupting such flows or decreasing the network connectivity can lead to a microtubule polarity reversal in spindles both in the simulations and in the experiments. Thus, we uncover an unexpected connection between spindle rheology and architecture in spindle self-organization.

scholarly journals The collapse of the spindle following ablation in S. pombe is mediated by microtubules and the motor protein dynein

2020 ◽  
Author(s):  
Parsa Zareiesfandabadi ◽  
Mary Williard Elting
Keyword(s):  
Laser Ablation ◽  
Molecular Motors ◽  
Mitotic Spindle ◽  
Motor Protein ◽  
Spindle Pole ◽  
Force Balance ◽  
Single Bundle ◽  
Spindle Pole Bodies ◽  
Basic Behavior ◽  
Supporting Force

AbstractA microtubule-based machine called the mitotic spindle segregates chromosomes when eukaryotic cells divide. In the fission yeast S. pombe, which undergoes closed mitosis, the spindle forms a single bundle of microtubules inside the nucleus. During elongation, the spindle extends via antiparallel microtubule sliding by molecular motors. These extensile forces from the spindle are thought to resist compressive forces from the nucleus. We probe the mechanism and maintenance of this force balance via laser ablation of spindles at various stages of mitosis. We find that spindle pole bodies collapse toward each other following ablation, but spindle geometry is often rescued, allowing spindles to resume elongation and segregate chromosomes. While this basic behavior has been previously observed, many questions remain about this phenomenon’s dynamics, mechanics, and molecular requirements. In this work, we find that previously hypothesized viscoelastic relaxation of the nucleus cannot fully explain spindle shortening in response to laser ablation. Instead, spindle collapse requires microtubule dynamics and is powered at least partly by the minus-end directed motor protein dynein. These results suggest a role for dynein in redundantly supporting force balance and bipolarity in the S. pombe spindle.

scholarly journals Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

2021 ◽  
Vol 22 (14) ◽  
pp. 7704
Author(s):  
Sayi’Mone Tati ◽  
Laleh Alisaraie
Keyword(s):  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Motor Protein ◽  
Structural Data ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Motor Domain ◽  
Conformational Search ◽  
Structural Mechanism

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

The projection domain of MAP2b regulates microtubule protrusion and process formation in Sf9 cells

2002 ◽  
Vol 115 (7) ◽  
pp. 1523-1539 ◽  
Author(s):  
Dave Bélanger ◽  
Carole Abi Farah ◽  
Minh Dang Nguyen ◽  
Michel Lauzon ◽  
Sylvie Cornibert ◽  
...  
Keyword(s):  
Cell Surface ◽  
Binding Domain ◽  
Intramolecular Interactions ◽  
Microtubule Assembly ◽  
Sf9 Cells ◽  
Microtubule Binding ◽  
Process Formation ◽  
Microtubule Binding Domain ◽  
Projection Domain

The expression of microtubule-associated protein 2 (MAP2), developmentally regulated by alternative splicing, coincides with neurite outgrowth. MAP2 proteins contain a microtubule-binding domain (C-terminal) that promotes microtubule assembly and a poorly characterized domain, the projection domain(N-terminal), extending at the surface of microtubules. MAP2b differs from MAP2c by an additional sequence of 1372 amino acids in the projection domain. In this study, we examined the role of the projection domain in the protrusion of microtubules from the cell surface and the subsequent process formation in Sf9 cells. In this system, MAP2b has a lower capacity to induce process formation than MAP2c. To investigate the role of the projection domain in this event, we expressed truncated forms of MAP2b and MAP2c that have partial or complete deletion of their projection domain in Sf9 cells. Our results indicate that process formation is induced by the microtubule-binding domain of these MAP2 proteins and is regulated by their projection domain. Furthermore, the microtubule-binding activity of MAP2b and MAP2c truncated forms as well as the structural properties of the microtubule bundles induced by them do not seem to be the only determinants that control the protrusion of microtubules from the cell surface in Sf9 cells. Rather, our data suggest that microtubule protrusion and process formation are regulated by intramolecular interactions between the projection domain and its microtubule-binding domain in MAP2b.

scholarly journals Inhibition of a Mitotic Motor Compromises the Formation of Dendrite-like Processes from Neuroblastoma Cells

1997 ◽  
Vol 136 (3) ◽  
pp. 659-668 ◽  
Author(s):  
Wenqian Yu ◽  
David J. Sharp ◽  
Ryoko Kuriyama ◽  
Prabhat Mallik ◽  
Peter W. Baas
Keyword(s):  
Retinoic Acid ◽  
Cell Division ◽  
Antisense Oligonucleotides ◽  
Motor Proteins ◽  
Molecular Motor ◽  
Neuroblastoma Cells ◽  
Opposite Orientation ◽  
Microtubule Polarity ◽  
Process Formation ◽  
Spindle Midzone

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.

Modeling Bidirectional Transport of New and Used Organelles in Fast Axonal Transport in Neurons

2010 ◽  
Vol 133 (1) ◽  
Author(s):  
A. V. Kuznetsov
Keyword(s):  
Axonal Transport ◽  
Cell Body ◽  
Molecular Motors ◽  
Molecular Motor ◽  
Fast Axonal Transport ◽  
Concentration Difference ◽  
Neuron Body ◽  
Microtubule Polarity ◽  
Bidirectional Transport ◽  
The Right

This paper develops a model for simulating transport of newly synthesized material from the neuron body toward the synapse of the axon as well as transport of misfolded and aggregated proteins back to the neuron body for recycling. The model demonstrates that motor-assisted transport, much similar to diffusion, can occur due to a simple concentration difference between the cell body and the synapse; organelles heading to the synapse do not need to attach preferably to plus-end-directed molecular motors, same as organelles heading to the neuron body for recycling do not need to attach preferably to minus-end-directed molecular motors. The underlying mechanics of molecular-motor-assisted transport is such that organelles would be transported to the right place even if new and used organelles had the same probability of attachment to plus-end-directed (and minus-end-directed) motors. It is also demonstrated that the axon with organelle traps and a region with a reversed microtubule polarity would support much smaller organelle fluxes of both new and used organelles than a healthy axon. The flux of organelles is shown to decrease as the width of organelle traps increases.

scholarly journals Mitotic Kinesin-Like Protein 2 Binds and Colocalizes with Papillomavirus E2 during Mitosis

2006 ◽  
Vol 81 (4) ◽  
pp. 1736-1745 ◽  
Author(s):  
Ting Yu ◽  
Yu-Cai Peng ◽  
Elliot J. Androphy
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Chromatin Immunoprecipitation ◽  
Viral Genome ◽  
Motor Protein ◽  
Dna Binding Protein ◽  
E2 Protein ◽  
Viral Genomes ◽  
Daughter Cells ◽  
Transcription And Replication

ABSTRACT MKlp2 is a kinesin-like motor protein of the central mitotic spindle required for completion of cytokinesis. Papillomavirus E2 is a sequence specific DNA binding protein that regulates viral transcription and replication and is responsible for partitioning viral episomes into daughter cells during cell division. We demonstrate that MKlp2 specifically associates with the E2 protein during mitosis. Using chromatin immunoprecipitation, we show viral genomes are in complex with MKlp2 only within this stage of cell cycle. By immunofluorescence, a subpopulation of papillomavirus E2 colocalizes with MKlp2 in the midbody/midplate during late mitosis. We conclude that during specific stages of mitosis, the papillomavirus E2 protein binds to MKlp2, and infer that association with this motor protein ensures viral genome partitioning during cytokinesis.

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