scholarly journals The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Tatyana Bodrug ◽  
Elizabeth M Wilson-Kubalek ◽  
Stanley Nithianantham ◽  
Alex F Thompson ◽  
April Alfieri ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Regulatory Mechanism ◽  
Atp Hydrolysis ◽  
Force Production ◽  
Bound State ◽  
Motor Domain ◽  
Tail Domain ◽  
Mitotic Spindles ◽  
Direct Binding ◽  
Mechanochemical Cycle

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.

scholarly journals The Kinesin-5 Tail Domain Directly Modulates the Mechanochemical Cycle of the Motor for Anti-Parallel Microtubule Sliding

2019 ◽  
Author(s):  
Tatyana Bodrug ◽  
Elizabeth Wilson-Kubalek ◽  
Stanley Nithianantham ◽  
Alex F. Thompson ◽  
April Alfieri ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Bound States ◽  
Atp Hydrolysis ◽  
Force Production ◽  
Tail Domain ◽  
Mitotic Spindles ◽  
Direct Binding ◽  
Biochemical Analyses ◽  
Mechanochemical Cycle

AbstractKinesin-5 motors organize mitotic spindles by sliding apart anti-parallel microtubules. They are homotetramers composed of two antiparallel dimers placing orthogonal motor and tail domains at opposite ends of a bipolar minifilament. Here, we describe a regulatory mechanism, involving direct binding of the tail to motor domain and reveal its fundamental role in microtubule sliding motility. Biochemical analyses reveal that the tail down-regulates microtubule-activated ATP hydrolysis by specifically engaging the motor in the nucleotide-free or ADP-bound states. Cryo-EM structures reveal that the tail stabilizes a unique conformation of the motor N-terminal subdomain opening its active site. Full-length kinesin-5 motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering along microtubules. The tail is critical for motors to zipper together two microtubules by generating substantial forces within sliding zones. The tail domain is essential for kinesin-5 mitotic spindle localization in vivo, which becomes severely reduced when the tail is deleted. Our studies suggest a revised microtubule-sliding model, in which tail domains directly engage motor domains at both ends of kinesin-5 homotetramers enhancing stability of the dual microtubule-bound states leading to slow motility yet high force production.

Clathrin in mitotic spindles

2000 ◽  
Vol 279 (2) ◽  
pp. C369-C374 ◽  
Author(s):  
Curtis T. Okamoto ◽  
Jeana McKinney ◽  
Young Y. Jeng
Keyword(s):  
Mitotic Spindle ◽  
Regulatory Mechanism ◽  
Staining Pattern ◽  
Brefeldin A ◽  
Polyclonal Antiserum ◽  
Mitotic Spindles ◽  
Madin Darby Canine Kidney ◽  
Western Blots ◽  
Darby Canine Kidney ◽  
Mitotic Cells

Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells.

scholarly journals Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Marvin E Tanenbaum ◽  
Ronald D Vale ◽  
Richard J McKenney
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Motor Proteins ◽  
Cytoplasmic Dynein ◽  
Eukaryotic Cells ◽  
Reverse Direction ◽  
Tail Domain ◽  
Mitotic Spindles

Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins.

Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules

2002 ◽  
Vol 115 (9) ◽  
pp. 1815-1824
Author(s):  
Laurence Haren ◽  
Andreas Merdes
Keyword(s):  
Cultured Cells ◽  
Direct Interaction ◽  
Sequence Motif ◽  
Tail Domain ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Direct Binding ◽  
Multiple Poles

In mitosis, NuMA localises to spindle poles where it contributes to the formation and maintenance of focussed microtubule arrays. Previous work has shown that NuMA is transported to the poles by dynein and dynactin. So far, it is unclear how NuMA accumulates at the spindle poles following transport and how it remains associated throughout mitosis. We show here that NuMA can bind to microtubules independently of dynein/dynactin. We characterise a 100-residue domain located within the C-terminal tail of NuMA that mediates a direct interaction with tubulin in vitro and that is necessary for NuMA association with tubulin in vivo. Moreover, this domain induces bundling and stabilisation of microtubules when expressed in cultured cells and leads to formation of abnormal mitotic spindles with increased microtubule asters or multiple poles. Our results suggest that NuMA organises the poles by stable crosslinking of the microtubule fibers.

scholarly journals Author response: The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding

2020 ◽  
Author(s):  
Tatyana Bodrug ◽  
Elizabeth M Wilson-Kubalek ◽  
Stanley Nithianantham ◽  
Alex F Thompson ◽  
April Alfieri ◽  
...  
Keyword(s):  
Motor Domain ◽  
Author Response ◽  
Tail Domain ◽  
Mechanochemical Cycle

3-D reconstruction and analysis of mammalian mitotic spindle structure

1992 ◽  
Vol 50 (1) ◽  
pp. 578-579
Author(s):  
Kent McDonald ◽  
David Mastronarde ◽  
Rubai Ding ◽  
Eileen O'Toole ◽  
J. Richard McIntosh
Keyword(s):  
Cross Sections ◽  
Mitotic Spindle ◽  
Experimental Studies ◽  
Video Camera ◽  
Three Dimensions ◽  
Mitotic Spindles ◽  
Black And White ◽  
Reconstruction Methods ◽  
Spindle Structure ◽  
Tissue Culture Line

Mammalian spindles are generally large and may contain over a thousand microtubules (MTs). For this reason they are difficult to reconstruct in three dimensions and many researchers have chosen to study the smaller and simpler spindles of lower eukaryotes. Nevertheless, the mammalian spindle is used for many experimental studies and it would be useful to know its detailed structure.We have been using serial cross sections and computer reconstruction methods to analyze MT distributions in mitotic spindles of PtK cells, a mammalian tissue culture line. Images from EM negatives are digtized on a light box by a Dage MTI video camera containing a black and white Saticon tube. The signal is digitized by a Parallax 1280 graphics device in a MicroVax III computer. Microtubules are digitized at a magnification such that each is 10-12 pixels in diameter.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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.

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