Microtubule choreography: spindle self-organization during cell division.

During cell division, the network of microtubules undergoes massive rearrangement to self-organize into the spindle, a bipolar structure essential for accurate chromosome segregation. This structure ensures the stable transmission of the genome from the mother cell to two daughter cells, yet the process by which the ordered architecture emerges from a collection of protein "parts" remains a mystery. In this review, we focus on several key spindle proteins, describing how they move, crosslink, and grow microtubules in vitro and contribute to the spindle's structural organization.

Quantitatively Analyzing the Morphological Growth Dynamics of Bipolar and Non-bipolar Spindles Using Xenopus Egg Extract.

The bipolar shape of the microtubule-based spindle is a pivotal morphological phenotype for accurate chromosome segregation during cell division. However, existing descriptions of spindle morphogenesis remain largely qualitative. Here, we introduce a method that provides a quantitative description of the morphological growth dynamics of spindles using Xenopus egg cytoplasmic extract and a computational image analysis pipeline.

HURP binding to the vinca domain of β-tubulin accounts for cancer drug resistance.

Vinca alkaloids, a class of tubulin-binding agent, are widely used in treating cancer, yet the emerging resistance compromises their efficacy. Hepatoma up-regulated protein (HURP), a microtubule-associated protein displaying heightened expression across various cancer types, reduces cancer cells' sensitivity to vinca-alkaloid drugs upon overexpression. However, the molecular basis behind this drug resistance remains unknown.

PIGB maintains nuclear lamina organization in skeletal muscle of Drosophila.

The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function.

Confined-microtubule assembly shapes three-dimensional cell wall structures in xylem vessels.

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood.

Morphological growth dynamics, mechanical stability, and active microtubule mechanics underlying spindle self-organization.

The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle's bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors.

Geometric trade-off between contractile force and viscous drag determines the actomyosin-based motility of a cell-sized droplet.

Cell migration in confined environments is fundamental for diverse biological processes from cancer invasion to leukocyte trafficking. The cell body is propelled by the contractile force of actomyosin networks transmitted from the cell membrane to the external substrates. However, physical determinants of actomyosin-based migration capacity in confined environments are not fully understood.

RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization.

Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear.

Local body weight measurement of the spindle.

The spindle is a micron-sized chromosome segregation machine built from microtubules and many other proteins. In this issue of Developmental Cell, Biswas et al. (2021) use sophisticated imaging and Xenopus egg extracts to show that the spindle's mass density is only as much as the surrounding cytoplasm, contrary to popular belief.

Nanoscopic changes in the lattice structure of striated muscle sarcomeres involved in the mechanism of spontaneous oscillatory contraction (SPOC).

Muscles perform a wide range of motile functions in animals. Among various types are skeletal and cardiac muscles, which exhibit a steady auto-oscillation of force and length when they are activated at an intermediate level of contraction. This phenomenon, termed spontaneous oscillatory contraction or SPOC, occurs devoid of cell membranes and at fixed concentrations of chemical substances, and is thus the property of the contractile system per se.

Mechanically Distinct Microtubule Arrays Determine the Length and Force Response of the Meiotic Spindle.

The microtubule-based spindle is subjected to various mechanical forces during cell division. How the structure generates and responds to forces while maintaining overall integrity is unknown because we have a poor understanding of the relationship between filament architecture and mechanics. Here, to fill this gap, we combine microneedle-based quantitative micromanipulation with high-resolution imaging, simultaneously analyzing forces and local filament motility in the Xenopus meiotic spindle.

Chromatin as a nuclear spring.

The nucleus in eukaryotic cells is the site for genomic functions such as RNA transcription, DNA replication, and DNA repair/recombination. However, the nucleus is subjected to various mechanical forces associated with diverse cellular activities, including contraction, migration, and adhesion. Although it has long been assumed that the lamina structure, underlying filamentous mesh-work of the nuclear envelope, plays an important role in resisting mechanical forces, the involvement of compact chromatin in mechanical resistance has also recently been suggested.

Analyzing the micromechanics of the cell division apparatus.

Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses.

Radial stiffness characteristics of the overlap regions of sarcomeres in isolated skeletal myofibrils in pre-force generating state.

We have studied the stiffness of myofilament lattice in sarcomeres in the pre-force generating state, which was realized by a relaxing reagent, BDM (butane dione monoxime). First, the radial stiffness for the overlap regions of sarcomeres of isolated single myofibrils was estimated from the resulting decreases in diameter by osmotic pressure applied with the addition of Dextran. Then, the radial stiffness was also estimated from force-distance curve measurements with AFM technology.

Regulation of mitotic spindle assembly factor NuMA by Importin-β.

Ran-guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-α/-β and spindle assembly factors (SAFs). The inhibition of SAFs performed by importins needs to be done without much sequestration from abundant nuclear localization signal (NLS) -containing proteins. However, the molecular mechanisms that determine NLS-binding selectivity and that inhibit activity of Importin-β-regulated SAFs (e.

High-quality frozen extracts of eggs reveal size-dependent control of metaphase spindle micromechanics.

Cell-free extracts from unfertilized eggs offer the opportunity for a variety of biochemical and biophysical assays for analyzing essential cell cycle events such as metaphase spindle assembly. However, the extracts often exhibit substantial variation in quality and have low storage stability, factors that hamper their experimental utility. Here we report a simple two-step method for preparing frozen egg extracts that retain spindle assembly activity levels similar to those of freshly prepared extracts.

Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow.

Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules.

The proper organization of the microtubule-based mitotic spindle is proposed to depend on nanometer-sized motor proteins generating forces that scale with a micron-sized geometric feature, such as microtubule overlap length. However, it is unclear whether such regulation can be achieved by any mitotic motor protein. Here, we employ an optical-trap- and total internal reflection fluorescence (TIRF)-based assay to show that ensembles of kinesin-5, a conserved mitotic motor protein, can push apart overlapping antiparallel microtubules to generate a force whose magnitude scales with filament overlap length.

Reconstitution of the augmin complex provides insights into its architecture and function.

Proper microtubule nucleation during cell division requires augmin, a microtubule-associated hetero-octameric protein complex. In current models, augmin recruits γ-tubulin, through the carboxyl terminus of its hDgt6 subunit to nucleate microtubules within spindles. However, augmin's biochemical complexity has restricted analysis of its structural organization and function.

Locally and globally coupled oscillators in muscle.

At an intermediate activation level, striated muscle exhibits autonomous oscillations called SPOC, in which the basic contractile units, sarcomeres, oscillate in length, and various oscillatory patterns such as traveling waves and their disrupted forms appear in a myofibril. Here we show that these patterns are reproduced by mechanically connecting in series the unit model that explains characteristics of SPOC at the single-sarcomere level. We further reduce the connected model to phase equations, revealing that the combination of local and global couplings is crucial to the emergence of these patterns.

Asymmetric friction of nonmotor MAPs can lead to their directional motion in active microtubule networks.

Diverse cellular processes require microtubules to be organized into distinct structures, such as asters or bundles. Within these dynamic motifs, microtubule-associated proteins (MAPs) are frequently under load, but how force modulates these proteins' function is poorly understood. Here, we combine optical trapping with TIRF-based microscopy to measure the force dependence of microtubule interaction for three nonmotor MAPs (NuMA, PRC1, and EB1) required for cell division.

Micromechanics of the vertebrate meiotic spindle examined by stretching along the pole-to-pole axis.

The meiotic spindle is a bipolar molecular machine that is designed to segregate duplicated chromosomes toward the opposite poles of the cell. The size and shape of the spindle are considered to be maintained by a balance of forces produced by molecular motors and microtubule assembly dynamics. Several studies have probed how mechanical perturbations of the force balance affect the spindle structure.

Using micromanipulation to analyze control of vertebrate meiotic spindle size.

The polymerization/depolymerization dynamics of microtubules (MTs) have been reported to contribute to control of the size and shape of spindles, but quantitative analysis of how the size and shape correlate with the amount and density of MTs in the spindle remains incomplete. Here, we measured these parameters using 3D microscopy of meiotic spindles that self-organized in Xenopus egg extracts and presented a simple equation describing the relationship among these parameters. To examine the validity of the equation, we cut the spindle into two fragments along the pole-to-pole axis by micromanipulation techniques that rapidly decrease the amount of MTs.