Microtubule-based Forces Research Articles

Force transmission through the inner kinetochore is enhanced by centromeric DNA sequences.

Previously, we reconstituted a minimal functional kinetochore from recombinant S. cerevisiae proteins that was capable of transmitting force from dynamic microtubules to nucleosomes containing the centromere-specific histone variant Cse4 (Hamilton et al. 2020). This work revealed two paths of force transmission through the inner kinetochore: through Mif2 and through the Okp1/Ame1 complex (OA). Here, using a chimeric DNA sequence that contains crucial centromere-determining elements of the budding yeast point centromere, we demonstrate that the presence of centromeric DNA sequences in Cse4-containing nucleosomes significantly strengthens OA-mediated linkages. Our findings indicate that centromeric sequences are important for the transmission of microtubule-based forces to the chromosome.

Dynamic interplay of microtubule and actomyosin forces drive tissue extension

In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. We show that wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, we confirm that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces.

Mechanisms underlying Myosin 10's contribution to the maintenance of mitotic spindle bipolarity.

Myosin 10 (Myo10) couples microtubules and integrin-based adhesions to movement along actin filaments via its microtubule-binding MyTH4 domain and integrin-binding FERM domain, respectively. Here we show that Myo10-depleted HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in Myo10-depleted MEFs and in Myo10-depleted HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates y-tubulin-positive acentriolar foci that serve as extra spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both microtubules and integrins to promote PCM/pole integrity. Conversely, Myo10 only needs interact with integrins to promote supernumerary centrosome clustering. Importantly, images of metaphase Halo-Myo10 knockin cells show that the myosin localizes exclusively to the spindle and the tips of adhesive retraction fibers. We conclude that Myo10 promotes PCM/pole integrity in part by interacting with spindle microtubules, and that it promotes supernumerary centrosome clustering by supporting retraction fiber-based cell adhesion, which likely serves to anchor the microtubule-based forces driving pole focusing.

Mechanobiology of the Mitotic Spindle.

The mitotic spindle is a microtubule-based assembly that separates the chromosomes during cell division. As the spindle is basically a mechanical micro machine, the understanding of its functioning is constantly motivating the development of experimental approaches based on mechanical perturbations, which are complementary to and work together with the classical genetics and biochemistry methods. Recent data emerging from these approaches in combination with theoretical modeling led to novel ideas and significant revisions of the basic concepts in the field. In this Perspective, we discuss the advances inthe understanding of spindle mechanics, focusing on microtubule forces that control chromosome movements.

Help or hindrance: how do microtubule-based forces contribute to genome damage and repair?

Forces generated by molecular motors and the cytoskeleton move the nucleus and genome during many cellular processes, including cell migration and division. How these forces impact the genome, and whether cells regulate cytoskeletal forces to preserve genome integrity is unclear. We recently demonstrated that, in budding yeast, mutants that stabilize the microtubule cytoskeleton cause excessive movement of the mitotic spindle and nucleus. We found that increased nuclear movement results in DNA damage and increased time to repair the damage through homology-directed repair. Our results indicate that nuclear movement impairs DNA repair through increased tension on chromosomes and nuclear deformation. However, the previous studies have shown genome mobility, driven by cytoskeleton-based forces, aids in homology-directed DNA repair. This sets up an apparent paradox, where genome mobility may prevent or promote DNA repair. Hence, this review explores how the genome is affected by nuclear movement and how genome mobility could aid or hinder homology-directed repair.

Resistance is futile: Centering forces yield for asymmetric cell division.

Asymmetric cell division relies on microtubule-based forces to asymmetrically position the mitotic apparatus. In this issue, Sallé et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807102) use magnetic tweezers to induce asymmetric division in sea urchin zygotes, demonstrating that asymmetry could arise from a time-dependent weakening of centering forces.

Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis.

Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis1,2. However, unlike for actin-based mechanical forces, our knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. Here, we establish microtubules as central components of cell mechanics during tissue morphogenesis. We find that individual cells are mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. We further establish that the Fat planar cell polarity (Ft-PCP) signalling pathway3,4 couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels.

Cytoskeletal mechanics of proplatelet maturation and platelet release

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. Although the mechanics of proplatelet elongation have been studied, the terminal steps of proplatelet maturation and platelet release remain poorly understood. To elucidate this process, released proplatelets were isolated, and their conversion into individual platelets was assessed. This enabled us to (a) define and quantify the different stages in platelet maturation, (b) identify a new intermediate stage in platelet production, the preplatelet, (c) delineate the cytoskeletal mechanics involved in preplatelet/proplatelet interconversion, and (d) model proplatelet fission and platelet release. Preplatelets are anucleate discoid particles 2-10 µm across that have the capacity to convert reversibly into elongated proplatelets by twisting microtubule-based forces that can be visualized in proplatelets expressing GFP-β1-tubulin. The release of platelets from the ends of proplatelets occurs at an increasing rate in time during culture, as larger proplatelets undergo successive fission, and is potentiated by shear.

Uncovering chromatin’s contribution to the mitotic spindle: Applications of computational and polymer models

The mitotic spindle is a structure that forms during mitosis to help ensure that each daughter cell receives a full complement of genetic material. In metaphase, the spindle contains microtubules that nucleate inward from two opposing poles. Chromosomes are attached to plus-ends of these microtubules via protein structures called kinetochores. The centromere is the specific region of kinetochore attachment on the chromosome. Chromatin surrounding the centromere (pericentric chromatin) is subject to microtubule-based forces and is commonly modeled as a linear spring, where the force that it exerts is proportional to the distance that it is stretched. We have incorporated physically based models of chromatin to create more accurate and predictive models of the spindle. In addition, using fluorescence microscopy and motion analysis of fluorescently labeled chromatin spots we discovered that pericentric chromatin is restrained relative to free diffusive motion. The characterization of chromatin is crucial to understand mitotic spindle stability and to understand the cell cycle checkpoint regulating anaphase onset.

Basal body stability and ciliogenesis requires the conserved component Poc1

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain-containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis.

Investigation of the Mechanistic Events Governing Platelet Release.

Abstract 4590Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions from which platelets release. While the mechanics of proplatelet elongation have been studied, the terminal steps of platelet production remain poorly understood. To understand the mechanics of platelet release, we developed a novel gradient sedimentation protocol to isolate released proplatelets. This has enabled us to define and quantify different stages in platelet maturation and identify a new intermediate stage in platelet production—the preplatelet. Preplatelets are anucleate discoid particles considerably larger (2-10μm) than platelets that have the capacity to reversibly convert into barbell-shaped proplatelets. Video-microscopy of GFP-tubulin expressing cells reveals that preplatelets elongate and twist into barbell forms by a process mediated by microtubule-based forces. α-Granules and dense granules present in the preplatelet translocate between platelet-sized swellings during barbell proplatelet formation, and ultimately concentrate at the terminal loops. Fission releases two mature platelets from the barbell ends, and isolated proplatelets will mature into bona fide platelets in culture over a 5-day period. Correspondingly, mature platelets are generated within 2 hours post-transfusion of fluorescently-tagged proplatelets into recipient mice. Released proplatelets were plated on extracellular matrix proteins (fibrinogen, laminin, fibronectin, collagen, matrigel) or a BSA control, and cultured for 24 hours to recapitulate the bone marrow vascular niche and determine whether these cell-substrate interactions mediate platelet release. Fibrinogen and laminin binding increased platelet release roughly 4-fold, suggesting cell-substrate interactions in the bone marrow vascular niche and surrounding vasculature are critical for the final stages or platelet production. These findings confirm that proplatelets mature into multiple individual platelets both in vivo/vitro, and begin to define the mechanistic events leading to platelet production. Disclosures:No relevant conflicts of interest to declare.

Cytoskeletal mechanisms for platelet production

Platelets release from megakaryocytes living primarily in the bone marrrow space. Intermediates in platelet formation are proplatelets, long tube-like processes that are extended by microtubule-based forces hundreds of micrometers from the megakaryocyte cell body. Granules and organelles enter the proplatelets and traffic up and down on their microtubules to be ultimately delivered to the platelet buds before they release. How individual platelets form and release from proplatelets remains poorly understood.

Mechanisms of organelle transport and capture along proplatelets during platelet production

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. Platelet packaging and release concludes at the tips of each proplatelet. Essential in this process is the distribution of organelles and platelet-specific granules into the nascent platelets. To investigate the mechanism of delivery of organelles into putative platelets, the distribution and dynamics of organelles/granules was monitored. Individual organelles are sent from the cell body to the proplatelets where they move bidirectionally until they are captured at proplatelet ends. Movement occurs at approximately 0.2 microm/min, but pauses and changes in direction are frequent. At any given time, approximately 30% of organelles/granules are in motion. Actin poisons do not diminish organelle motion, and vesicular structures are intimately associated with the microtubules. Therefore, movement appears to involve microtubule-based forces. Bidirectional organelle movement is conveyed by the bipolar organization of microtubules within the proplatelet, as kinesin-coated beads move bidirectionally on the microtubule arrays of permeabilized proplatelets. Movement of organelles along proplatelets involves 2 mechanisms: organelles travel along microtubules, and the linked microtubules move relative to each other. These studies demonstrate that the components that form platelets are delivered to and assembled de novo along proplatelets.

Actin-myosin–based contraction is responsible for apoptotic nuclear disintegration

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

The birth of the platelet

Platelets are small subcellular fragments that are formed from the cytoplasm of bone marrow megakaryocytes, which circulate in blood with characteristic discoid shapes. To assemble and release platelets, megakaryocytes follow a maturation program that accumulates in the conversion of the bulk of their cytoplasmic into multiple long processes called proplatelets. A megakaryocyte may protrude as many as 10-20 proplatelets, each which begins as a blunt protrusion that is driven out by microtubule-based forces. With time, these protrusions thin and branch repeatedly. Platelets form only at the ends of proplatelets. As the nascent platelet matures, its content of granules and organelles are delivered as a stream of individual particles moving from the megakaryocyte cell body to the proplatelet tip. Once the platelet has been filled with its content of intracellular materials, a single microtubule approximately 100 micro m in length is rolled into a coil, and the platelet releases into the medium. Platelet formation can be divided into two phases. In the first phase, there is nuclear proliferation to 16-32xN and the enlargement of the megakaryocyte cytoplasm as it is filled with cytoskeletal proteins, platelet specific granules and granule contents and membranous systems. This phase occurs over a period of days and requires induction by megakaryocyte specific growth factors. Proplatelets are extended in the second phase and platelets are released. This phase is completed in hours.

A KID's game of tug o’ war

A hallmark of mitotic chromosome segregation is the aligning of chromosomes during metaphase. In order to position the duplicated genome at the ‘metaphase plate’, dividing cells utilize microtubule-based forces to push the chromosomes towards their central regions. The forces required to establish this arrangement of chromosomes at metaphase are the subject of two papers by Antonio et al.1xXkid, a chromokinesin required for chromosome alignment on the metaphase plate. Antonio, C. et al. Cell. 2000; 102: 425–435Abstract | Full Text | Full Text PDF | PubMedSee all References1 and Funabiki and Murray2xThe Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement. Funabiki, H. and Murray, A.W. Cell. 2000; 102: 411–424Abstract | Full Text | Full Text PDF | PubMedSee all References2. Therein, the authors describe the role of the kinesin-like protein Xkid in generating the force responsible for chromosome congression during metaphase.Kid-like proteins were originally identified in Drosophila melanogaster in a screen for genes required for proper chromosome segregation during gametogenesis. One mutant gene, nod, encodes a protein with homology to the kinesin family of molecular motors. Later, a mammalian homologue, Kid (nesin-like ḎNA-binding protein), was discovered in a human carcinoma cell line. Consistent with a role in chromosome segregation, the human Kid protein was shown to localize along the length of chromosomes during M phase.The recent studies by these groups have further elucidated the mechanism by which Kid proteins contribute to chromosome segregation. Both groups cloned the Xenopus laevis homologue of Kid (Xkid), and, using the Xenopus egg extract system, they have shown that loss of Kid function leads to misalignment of chromosomes on the mitotic spindle during metaphase. Interestingly, this misalignment was due to the inability of chromosome arms to maintain their position at the metaphase plate: when antibodies against Xkid were added to metaphase-arrested extracts in which chromosomes had already aligned at the centre of the spindle, chromosome arms moved towards the poles independently of anaphase chromosome segregation.One consequence of the function of Xkid in maintaining chromosome position during metaphase is that it needs to be inactivated prior to anaphase so that chromosome segregation to the spindle poles can occur. In fact, Xkid is degraded by the anaphase-promoting complex/proteasome system at the onset of anaphase. Moreover, when a nondegradable form of Xkid was added to extracts, anaphase chromosome movement was inhibited.The findings in these two papers highlight several interesting aspects of mitotic spindle structure and function. Firstly, chromosome alignment during metaphase is not a static phenomenon; rather, it involves a balance of forces that act to push chromosomes towards the equator and pull them toward the poles. Secondly, the trigger for anaphase might actually be deactivation of the equatorial force, rather than the de novo activation of a poleward force. As such, the characterization of the Xkid motor sheds some light upon the process by which chromosomes undergo mitotic segregation in higher eukaryotes.