Cortical Force Generators Research Articles

Structural and functional insights into activation and regulation of the dynein-dynactin-NuMA complex.

During cell division, NuMA orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein motors, microtubules, and other cellular factors. Here we used in vitro reconstitution, cryo-electron microscopy, and live cell imaging to understand the mechanism and regulation of NuMA. We determined the structure of the processive dynein/dynactin/NuMA complex (DDN) and showed that the NuMA N-terminus drives dynein motility in vitro and facilitates dynein-mediated transport in live cells. The C-terminus of NuMA directly binds to and suppresses the dynamics of the microtubule minus-end. Full-length NuMA is autoinhibited, but mitotically phosphorylated NuMA activates dynein in vitro and interphase cells. Together with dynein, activated full-length NuMA focuses microtubule minus-ends into aster-like structures. The binding of the cortical protein LGN to the NuMA C-terminus results in preferential binding of NuMA to the microtubule plus-end. These results provide critical insights into the activation of NuMA and dynein for their functions in the spindle body and the cell cortex.

Relaxation and Noise-Driven Oscillations in a Model of Mitotic Spindle Dynamics

During cell division, the mitotic spindle moves dynamically through the cell to position the chromosomes and determine the ultimate spatial position of the two daughter cells. These movements have been attributed to the action of cortical force generators which pull on the astral microtubules to position the spindle, as well as pushing events by these same microtubules against the cell cortex and plasma membrane. Attachment and detachment of cortical force generators working antagonistically against centring forces of microtubules have been modelled previously (Grill et al. in Phys Rev Lett 94:108104, 2005) via stochastic simulations and mean-field Fokker–Planck equations (describing random motion of force generators) to predict oscillations of a spindle pole in one spatial dimension. Using systematic asymptotic methods, we reduce the Fokker–Planck system to a set of ordinary differential equations (ODEs), consistent with a set proposed by Grill et al., which can provide accurate predictions of the conditions for the Fokker–Planck system to exhibit oscillations. In the limit of small restoring forces, we derive an algebraic prediction of the amplitude of spindle-pole oscillations and demonstrate the relaxation structure of nonlinear oscillations. We also show how noise-induced oscillations can arise in stochastic simulations for conditions in which the mean-field Fokker–Planck system predicts stability, but for which the period can be estimated directly by the ODE model and the amplitude by a related stochastic differential equation that incorporates random binding kinetics.

Reduction of cortical pulling at mitotic entry facilitates aster centration.

Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from ∼300pN/µm to ∼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.

Model-based trajectory classification of anchored molecular motor-biopolymer interactions

During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units in vivo. There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in C. elegans zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA).

PP2A--B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA.

Proper orientation of the mitotic spindle is critical for accurate development and morphogenesis. In human cells, spindle orientation is regulated by the evolutionarily conserved protein NuMA, which interacts with dynein and enriches it at the cell cortex. Pulling forces generated by cortical dynein orient the mitotic spindle. Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) negatively regulates its cortical localization. Thus, only NuMA not phosphorylated at T2055 localizes at the cell cortex. However, the identity and the mechanism of action of the phosphatase complex involved in T2055 dephosphorylation remains elusive. Here, we characterized the PPP2CA-B55γ (PPP2R2C)-PPP2R1B complex that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle orientation. In vitro reconstitution experiments revealed that this complex is sufficient for T2055 dephosphorylation. Importantly, we identified polybasic residues in NuMA that are critical for T2055 dephosphorylation, and for maintaining appropriate cortical NuMA levels for accurate spindle elongation. Furthermore, we found that Cdk1-mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation at T2055 are reversible events. Altogether, this study uncovers a novel mechanism by which Cdk1 and its counteracting PP2A-B55γ complex orchestrate spatiotemporal levels of cortical force generators for flawless mitosis.

Hexameric NuMA:LGN structures promote multivalent interactions required for planar epithelial divisions

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.

Numerical study on spindle positioning using phase field method

A method of numerical simulation of cell division using phase fields is presented. The cell division plane is obtained as a result of the spindle position and orientation considered with the spatial distribution of the activated cortical force generators and the dividing cell shape. To exemplify the application of the proposed method, numerical simulations of the development of cysts and early embryos are performed. The numerical results demonstrate that the activated cortical force generators that are localized at the lateral cortices of the epithelial cells lead to the formation of a single central lumen. It is additionally shown that the linear distribution of the activated cortical force generators along the animal-vegetal axis of a spherical cell engenders a similar cell proliferation of the divided embryo generated by the 32 cell period in a sea cucumber.

Direct measurement of cortical force generation and polarization in a living parasite.

Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria, and cryptosporidiosis. A conserved motile system is critical to their life cycles and drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality but becomes directed toward the rear of the cell after a period of time. The transition from nondirectional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens.

Regulation of mitotic spindle orientation: an integrated view.

Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gαi/LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGN-independent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation.

Need for multi-scale systems to identify spindle orientation regulators relevant to tissue disorganization in solid cancers.

OPINION article Front. Physiol., 25 July 2014Sec. Systems Biology Archive https://doi.org/10.3389/fphys.2014.00278

General theory for the mechanics of confined microtubule asters

In cells, dynamic microtubules organize into asters or spindles to assist positioning of organelles. Two types of forces are suggested to contribute to the positioning process: (i) microtubule-growth based pushing forces; and (ii) motor protein mediated pulling forces. In this paper, we present a general theory to account for aster positioning in a confinement of arbitrary shape. The theory takes account of microtubule nucleation, growth, catastrophe, slipping, as well as interaction with cortical force generators. We calculate microtubule distributions and forces acting on microtubule organizing centers in a sphere and in an ellipsoid. Positioning mechanisms based on both pushing forces and pulling forces can be distinguished in our theory for different parameter regimes or in different geometries. In addition, we investigate positioning of microtubule asters in the case of asymmetric distribution of motors. This analysis enables us to characterize situations relevant for Caenorrhabditis elegans embryos.

G protein regulator 1 (GPR‐1) localizes to cortical sites of artificial mechanical indentation in Caenorhabditis elegans zygotes

Cytokinesis and spindle positioning require the cortical force regulator G Protein Regulator 1/2 (GPR-1/2). GPR-1/2 is thought to localize to sites of cortical force generation. Does GPR-1/2 also act as a sensor for mechanical stimulation? I mechanically stimulated the cortex by indenting it with a glass needle and observed the cortical localization of a YFP::GPR-1 transgene. I found that cortical YFP::GPR-1 accumulated at the site of mechanical indentation. This phenomenon occurred on most of the cortical areas except the site of prospective cytokinesis furrow formation. This result suggests that GPR-1/2 can sense mechanical properties of the cortex, which may be important for GPR-1/2 function regulating spindle positioning and cytokinesis.

Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation

Mitotic spindle positioning by cortical pulling forces1 defines the cell division axis and location2, which is critical for proper cell division and development3. Although recent work has identified developmental and extrinsic cues that regulate spindle orientation4-6, the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle pole and chromosome-derived signals that regulate cytoplasmic dynein localization. First, dynein displays a dynamic asymmetric cortical localization that is negatively regulated by spindle pole proximity resulting in spindle oscillations to center the spindle within the cell. We find that this signal is comprised of the spindle pole localized Polo-like kinase (Plk1), which regulates dynein localization by controlling the interaction between dynein-dynactin and its upstream cortical targeting factors NuMA and LGN. Second, a chromosome-derived Ran-GTP gradient restricts the localization of NuMA-LGN to the lateral cell cortex to define and maintain the spindle orientation axis. Ran-GTP acts in part through NuMA’s nuclear localization sequence to locally alter the ability of NuMA-LGN to associate with the cell cortex in the vicinity of chromosomes. We propose that these chromosome and spindle pole-derived gradients generate an intrinsic code to control spindle position and orientation.

The Forces that Center the Mitotic Spindle

Precise positioning of the mitotic spindle is important for specifying the plane of cell division and the subsequent partitioning of the cell's contents to the daughter cells. Studies on different organisms and cell types have suggested diverse centering mechanisms: astral microtubules grow out from the spindle and push against the cortex, cortical dynein motors pull on astral microtubules, and dynein-dependent organelle transport on astral microtubules leads to a reactive force on the spindle. The different mechanisms lead to different predictions for the precision of centering, how mutations effect the precision, and the magnitude of the forces associated with spindle centering. We used image processing to accurately track the position and orientation of the mitotic spindle during the first cell division in the C. elegans embryo. The high precision of centering, < 1% of cell diameter transverse to the anterior-posterior axis, increased after RNAi against gpr-1/2, genes encoding activators of the cortical force generators; this suggests that centering is not mediated by gpr-1/2-dependent cortical pulling forces. To measure the forces associated with spindle positioning, we built a magnetic tweezers apparatus so that forces could be exerted on the spindle via beads incorporated into the embryo: forces of approximately 20 pN were required to displace the spindle through 1 μm. These mechanical experiments constrain molecular models of the centering process.

Membrane Invaginations Reveal Cortical Sites that Pull on Mitotic Spindles in One-Cell C. elegans Embryos

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell.

Role of cortical rigidity in spindle positioning in C. elegans

The first cell division in C. elegans is asymmetric. Asymmetric cell division requires correct positioning of the mitotic spindle. Prior to metaphase, the nuclear-centrosome complex, the precursor of the mitotic spindle, is positioned in the cell center. During anaphase, the spindle is displaced towards the posterior so that bisection of the spindle during cytokinesis leads to daughter cells of unequal sizes. Forces that center and position the spindle come from cortical force generators that pull on astral microtubules. In order to generate force, the cortex needs to provide a stiff anchoring platform. However, a role for the cortex in C. elegans has only been described with respect to polarity establishment. We perturbed the acto-myosin cortex by RNAi of non-muscle-myosin II (nmy-2) using conditions that allowed us to avoid disturbing polarity. Strikingly, in nmy-2(RNAi), membrane tubes are pulled from the plasma membrane into the cell. They were seen after RNAi against other actin cytoskeleton proteins and members of force generation complex, suggesting that the cortical force generators pull the invaginations, and a weakening of the cortex. As expected, we observed an increase in the variance of spindle position and orientation in nmy-2(RNAi). We used the oscillations of the centrosomes during anaphase as a reporter of spindle mechanics, and measured an increase in oscillations frequency but only a marginal decrease in amplitude. In order to understand this phenotype, we used our previously published model to analyze the results. Only by including the cortex into the model, we were able to fully describe the role of NMY-2. In summary, the occurrence of tubes after nmy-2(RNAi) strongly points towards a weakening of the cortex and the analysis of the spindle positioning suggests that the cortex provides a rigid platform for anchoring the force generators.

Physical Description of Mitotic Spindle Orientation During Cell Division

During cell division, the duplicated chromosomes are physically separated by the action of the mitotic spindle. The mitotic spindle is a dynamic structure of the cytoskeleton, which consists of two microtubule asters. Its orientation defines the axis along which the cell divides. Recent experiments on dividing cells, which adhere to patterned substrates, show that the spindle orientation depends on the spatial distribution of cell adhesion sites. In the present work we show that the experimentally observed spindle orientation can be understood as the result of the action of cortical force generators acting on the spindle microtubules. We assume that the local activity of force generators is controlled by the spatial distribution of cell adhesion sites determined by the particular geometry of the adhesive substrate. We develop a simple physical description of the spindle mechanics, which allows us to calculate the torque acting on the spindle, as well as the energy profile and the angular distribution of spindle orientation. Our model accounts for the preferred spindle orientation, as well as the full shape of the angular distributions of spindle orientation observed in a large variety of pattern geometries. Remarkably, it also describes the transition from symmetric to asymmetric spindle orientation, observed for certain changes of the shape of the adhesive patterns. We conclude that, on the basis of a few simple assumptions, we can provide a quantitative description of the spindle orientation of adherent cells. M. Thery, A. Jimenez-Dalmaroni, et al., Nature 447, 493 (2007).

Dynamic localization of LIN-5 and GPR-1/2 to cortical force generation domains during spindle positioning

G protein signaling pathways regulate mitotic spindle positioning during cell division in many systems. In Caenorhabditis elegans embryos, Gα subunits act with the positive regulators GPR-1/2 and LIN-5 to generate cortical pulling forces for posterior spindle displacement during the first asymmetric division. GPR-1/2 are asymmetrically localized at the posterior cortex by PAR polarity cues at this time. Here we show that LIN-5 colocalizes with GPR-1/2 in one-cell embryos during spindle displacement. Significantly, we also find that LIN-5 and GPR-1/2 are localized to the opposite, anterior cortex in a polarity-dependent manner during the nuclear centration and rotation movements that orient the forming spindle onto the polarity axis. The depletion of LIN-5 or GPR-1/2 results in decreased centration and rotation rates, indicating a role in force generation at this stage. The localization of LIN-5 and GPR-1/2 is largely interdependent and requires Gα. Further, LIN-5 immunoprecipitates with Gα in vivo, and this association is GPR-1/2 dependent. These results suggest that a complex of Gα/GPR-1/2/LIN-5 is asymmetrically localized in response to polarity cues, and this may be the active signaling complex that transmits asymmetries to the force generation machinery during both nuclear rotation and spindle displacement.

Coupling of cortical dynein and Gα proteins mediates spindle positioning in Caenorhabditis elegans

Despite being essential for spatial cell division control, the mechanisms governing spindle positioning remain incompletely understood. In the Caenorhabditis elegans one-cell stage embryo, the spindle becomes asymmetrically positioned during anaphase through the action of as-yet unidentified cortical force generators that pull on astral microtubules and that depend on two G alpha proteins and associated proteins. We performed spindle-severing experiments following temporally restricted gene inactivation and drug exposure, and established that microtubule dynamics and dynein are both required for generating efficient pulling forces. We found that the G alpha-associated proteins GPR-1/2 and LIN-5 interact in vivo with LIS-1, a component of the dynein complex. Moreover, we discovered that the LIN-5, GPR-1/2 and the G alpha proteins promote the presence of the dynein complex at the cell cortex. Our findings suggest a mechanism by which the G alpha proteins enable GPR-1/2 and LIN-5 recruitment to the cortex, thus ensuring the presence of cortical dynein. Together with microtubule dynamics, this allows pulling forces to be exerted and proper cell division to be achieved.

Experimental and theoretical study of mitotic spindle orientation

The architecture and adhesiveness of a cell microenvironment is a critical factor for the regulation of spindle orientation in vivo. Using a combination of theory and experiments, we have investigated spindle orientation in HeLa (human) cells. Here we show that spindle orientation can be understood as the result of the action of cortical force generators, which interact with spindle microtubules and are activated by cortical cues. We develop a simple physical description of this spindle mechanics, which allows us to calculate angular profiles of the torque acting on the spindle, as well as the angular distribution of spindle orientations. Our model accounts for the preferred spindle orientation and the shape of the full angular distribution of spindle orientations observed in a large variety of different cellular microenvironment geometries. It also correctly describes asymmetric spindle orientations, which are observed for certain distributions of cortical cues. We conclude that, on the basis of a few simple assumptions, we can provide a quantitative description of the spindle orientation of adherent cells.