A new model for binding of kinesin 13 to curved microtubule protofilaments

Kinesin motor proteins use adenosine triphosphate hydrolysis to do work on microtubules (MTs). Most kinesins walk along the MT, but class 13 kinesins instead uniquely recognize MT ends and depolymerize MT protofilaments. We have used electron microscopy (EM) to understand the molecular interactions by which kinesin 13 performs these tasks. Although a construct of only the motor domain of kinesin 13 binds to every heterodimer of a tubulin ring, a construct containing the neck and the motor domain occupies alternate binding sites. Likewise, EM maps of the dimeric full-length (FL) protein exhibit alternate site binding but reveal density for only one of two motor heads. These results indicate that the second head of dimeric kinesin 13 does not have access to adjacent binding sites on the curved protofilament and suggest that the neck alone is sufficient to obstruct access. Additionally, the FL construct promotes increased stacking of rings compared with other constructs. Together, these data suggest a model for kinesin 13 depolymerization in which increased efficiency is achieved by binding of one kinesin 13 molecule to adjacent protofilaments.

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The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry

eLife ◽

10.7554/elife.27793 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 20

Author(s):

Joseph Atherton ◽

I-Mei Yu ◽

Alexander Cook ◽

Joseph M Muretta ◽

Agnel Joseph ◽

...

Keyword(s):

Electron Microscopy ◽

Motor Function ◽

Nucleotide Binding ◽

Motor Domain ◽

Kinesin Motor ◽

Binding Motifs ◽

Cryo Electron Microscopy ◽

Specific Sequences

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.

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Kinesin-13s form rings around microtubules

The Journal of Cell Biology ◽

10.1083/jcb.200605194 ◽

2006 ◽

Vol 175 (1) ◽

pp. 25-31 ◽

Cited By ~ 43

Author(s):

Dongyan Tan ◽

Ana B. Asenjo ◽

Vito Mennella ◽

David J. Sharp ◽

Hernando Sosa

Keyword(s):

Electron Microscopy ◽

Adenosine Triphosphate ◽

Chromosome Segregation ◽

Motor Proteins ◽

General Description ◽

Oligomeric Structure ◽

First Report ◽

Family Form

Kinesin is a superfamily of motor proteins that uses the energy of adenosine triphosphate hydrolysis to move and generate force along microtubules. A notable exception to this general description is found in the kinesin-13 family that actively depolymerizes microtubules rather than actively moving along them. This depolymerization activity is important in mitosis during chromosome segregation. It is still not fully clear by which mechanism kinesin-13s depolymerize microtubules. To address this issue, we used electron microscopy to investigate the interaction of kinesin-13s with microtubules. Surprisingly, we found that proteins of the kinesin-13 family form rings and spirals around microtubules. This is the first report of this type of oligomeric structure for any kinesin protein. These rings may allow kinesin-13s to stay at the ends of microtubules during depolymerization.

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Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1811 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1811-1820 ◽

Cited By ~ 113

Author(s):

V I Rodionov ◽

F K Gyoeva ◽

E Tanaka ◽

A D Bershadsky ◽

J M Vasiliev ◽

...

Keyword(s):

Cell Shape ◽

Motor Proteins ◽

Critical Role ◽

Leading Edge ◽

Transport Processes ◽

Motor Domain ◽

Organelle Transport ◽

Limited Area ◽

Kinesin Motor ◽

Cytoplasmic Microtubules

One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.

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Sites of Insulin Action Using Ferritin Labeling

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066759 ◽

1971 ◽

Vol 29 ◽

pp. 540-541

Author(s):

Burton B. Silver ◽

Ronald S. Nelson

Keyword(s):

Electron Microscopy ◽

Binding Sites ◽

Anterior Pituitary ◽

Insulin Action ◽

Diabetic Rats ◽

Insulin Antibody ◽

Fat Pad ◽

Target Organs ◽

Uranium Salts ◽

Sugar Levels

Some investigators feel that insulin does not enter cells but exerts its influence in some manner on the cell surface. Ferritin labeling of insulin and insulin antibody was used to determine if binding sites of insulin to specific target organs could be seen with electron microscopy.Alloxanized rats were considered diabetic if blood sugar levels were in excess of 300 mg %. Test reagents included ferritin, ferritin labeled insulin, and ferritin labeled insulin antibody. Target organs examined were were diaphragm, kidney, gastrocnemius, fat pad, liver and anterior pituitary. Reagents were administered through the left common carotid. Survival time was at least one hour in test animals. Tissue incubation studies were also done in normal as well as diabetic rats. Specimens were fixed in gluteraldehyde and osmium followed by staining with lead and uranium salts. Some tissues were not stained.

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Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)31692-2 ◽

1994 ◽

Vol 269 (51) ◽

pp. 32708

Author(s):

T G Huang ◽

J Suhan ◽

D D Hackney

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Position ◽

Motor Domain ◽

Kinesin Motor

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Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00351.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. G328-G339 ◽

Cited By ~ 41

Author(s):

P. Singh ◽

X. Lu ◽

S. Cobb ◽

B. T. Miller ◽

N. Tarasova ◽

...

Keyword(s):

Epithelial Cells ◽

Binding Sites ◽

Intestinal Epithelial Cells ◽

Full Length ◽

Intestinal Epithelial ◽

Growth Effects ◽

High Affinity ◽

Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

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How kinesin motor proteins drive mitotic spindle function: Lessons from molecular assays

Seminars in Cell and Developmental Biology ◽

10.1016/j.semcdb.2010.01.018 ◽

2010 ◽

Vol 21 (3) ◽

pp. 260-268 ◽

Cited By ~ 84

Author(s):

Linda Wordeman

Keyword(s):

Mitotic Spindle ◽

Motor Proteins ◽

Kinesin Motor ◽

Molecular Assays ◽

Kinesin Motor Proteins ◽

Spindle Function

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The mechanism of kinesin inhibition by kinesin-binding protein

eLife ◽

10.7554/elife.61481 ◽

2020 ◽

Vol 9 ◽

Author(s):

Joseph Atherton ◽

Jessica JA Hummel ◽

Natacha Olieric ◽

Julia Locke ◽

Alejandro Peña ◽

...

Keyword(s):

Cell Function ◽

Binding Protein ◽

Eukaryotic Cell ◽

Motor Domain ◽

Tetratricopeptide Repeat ◽

Kinesin Motor ◽

Temporal Regulation ◽

Cryo Electron Microscopy ◽

Local Environments ◽

Binding Surface

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP–kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain’s tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.

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