scholarly journals The mitotic spindle is chiral due to torques generated by motor proteins

2017 ◽  
Author(s):  
Maja Novak ◽  
Bruno Polak ◽  
Juraj Simunić ◽  
Zvonimir Boban ◽  
Barbara Kuzmić ◽  
...  
Keyword(s):  
Theoretical Model ◽  
Mitotic Spindle ◽  
Motor Proteins ◽  
Metaphase Plate ◽  
Super Resolution ◽  
Left Handed ◽  
Associated Proteins ◽  
Balance Of Forces ◽  
Micro Machine ◽  
Super Resolution Microscopy

AbstractMitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins1,2. Forces are required for the congression of chromosomes to the metaphase plate and separation of chromatids in anaphase3-6. However, torques may also exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles follow a left-handed helical path, which cannot be explained by forces but rather by torques acting in the bundles. STED super-resolution microscopy, as well as confocal microscopy, of human spindles shows that the bundles have complex curved shapes. The average helicity of the bundles with respect to the spindle axis is 1.2°/μm. Inactivation of kinesin-5 (Eg5/Kif11) abolished the chirality of the spindle, suggesting that this motor generates the helical shape of microtubule bundles. To explain the observed shapes, we introduce a theoretical model for the balance of forces and torques acting in the spindle, and show that torque is required to generate the helical shapes. We conclude that torques generated by motor proteins, in addition to forces, exist in the spindle and determine its architecture.

scholarly journals Twist of the mitotic spindle culminates at anaphase onset and depends on microtubule-associated proteins along with external forces

2020 ◽  
Author(s):  
Monika Trupinić ◽  
Ivana Ponjavić ◽  
Barbara Kokanović ◽  
Ivan Barišić ◽  
Siniša Šegvić ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Molecular Mechanisms ◽  
Mechanical Response ◽  
Torsional Rigidity ◽  
Physiological Role ◽  
Cancer Cell Line ◽  
Left Handed ◽  
Associated Proteins ◽  
Bridging Fibers ◽  
External Forces

ABSTRACTMechanical forces produced by motor proteins and microtubule dynamics within the mitotic spindle are crucial for the movement of chromosomes and their segregation into the emerging daughter cells. In addition to linear forces, rotational forces are present in the spindle, reflected in the left-handed twisted shapes of microtubule bundles that make the spindle chiral. However, the molecular origins of spindle chirality are unknown. Here we show that spindles are most twisted at the beginning of anaphase, and reveal multiple molecular players involved in spindle chirality. Inhibition of Eg5/kinesin-5 in a non-cancer cell line abolished spindle twist and depletion of Kif18A/kinesin-8 resulted in a right-handed twist, implying that these motors regulate twist likely by rotating the microtubules around one another within the antiparallel overlaps of bridging fibers. Depletion of the crosslinker PRC1 resulted in a right-handed twist, indicating that PRC1 may contribute to the twist by constraining free rotation of microtubules. Overexpression of PRC1 abolished twist, possibly due to increased torsional rigidity of the bundles. Depletion of augmin led to a right-handed twist, suggesting that twist depends on the geometry of microtubule nucleation. Round spindles were more twisted than elongated ones, a notion that we directly tested by compressing the spindle along its axis, which resulted in stronger left-handed twist, indicating a correlation between bending moments and twist. We conclude that spindle twist is controlled by multiple molecular mechanisms acting at different locations within the spindle as well as forces, and propose a potential physiological role of twist in promoting passive mechanical response of the spindle to forces during metaphase.

scholarly journals Subcellular drug targeting illuminates local kinase action

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Paula J Bucko ◽  
Chloe K Lombard ◽  
Lindsay Rathbun ◽  
Irvin Garcia ◽  
Akansha Bhat ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Super Resolution ◽  
Biological Processes ◽  
Anchoring Proteins ◽  
Intracellular Compartments ◽  
Mitotic Spindle Assembly ◽  
Targeted Inhibition ◽  
Super Resolution Microscopy

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

scholarly journals Left-handed DNA-PAINT for improved superresolution imaging in the nucleus

2020 ◽  
Author(s):  
H.J. Geertsema ◽  
G. Aimola ◽  
V. Fabricius ◽  
J.P. Fuerste ◽  
B.B. Kaufer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Hybridization ◽  
Super Resolution ◽  
Dna Oligomers ◽  
Superresolution Imaging ◽  
Left Handed ◽  
Nanoscale Topography ◽  
Target Molecules ◽  
Cellular Dna ◽  
Super Resolution Microscopy

AbstractDNA point accumulation in nanoscale topography (DNA-PAINT) advances super-resolution microscopy with superior resolution and multiplexing capabilities. However, cellular DNA may interfere with this single-molecule localization technique based on DNA-DNA hybridization. Here, we introduce left-handed DNA (L-DNA) oligomers that do not hybridize to naturally present R-DNA and demonstrate that L-DNA PAINT has the same specificity and multiplexing capability as R-DNA PAINT, but greatly improves specific visualization of nuclear target molecules.

scholarly journals PPP1R35 is a novel centrosomal protein that regulates centriole length in concert with the microcephaly protein RTTN

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Andrew Michael Sydor ◽  
Etienne Coyaud ◽  
Cristina Rovelli ◽  
Estelle Laurent ◽  
Helen Liu ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Cellular Level ◽  
Mammalian Development ◽  
Centrosomal Protein ◽  
Associated Proteins ◽  
Bona Fide ◽  
Distal Centriole ◽  
Super Resolution Microscopy

Centrosome structure, function, and number are finely regulated at the cellular level to ensure normal mammalian development. Here, we characterize PPP1R35 as a novel bona fide centrosomal protein and demonstrate that it is critical for centriole elongation. Using quantitative super-resolution microscopy mapping and live-cell imaging we show that PPP1R35 is a resident centrosomal protein located in the proximal lumen above the cartwheel, a region of the centriole that has eluded detailed characterization. Loss of PPP1R35 function results in decreased centrosome number and shortened centrioles that lack centriolar distal and microtubule wall associated proteins required for centriole elongation. We further demonstrate that PPP1R35 acts downstream of, and forms a complex with, RTTN, a microcephaly protein required for distal centriole elongation. Altogether, our study identifies a novel step in the centriole elongation pathway centered on PPP1R35 and elucidates downstream partners of the microcephaly protein RTTN.

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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