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Rapid and reductive cell divisions during embryogenesis require that intracellular structures adapt to a wide range of cell sizes. The mitotic spindle presents a central example of this flexibility, scaling with the dimensions of the cell to mediate accurate chromosome segregation. To determine whether spindle size regulation is achieved through a developmental program or is intrinsically specified by cell size or shape, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes. Spindle size was observed to shrink with decreasing compartment size, similar to what occurs during early embryogenesis, and this scaling trend depended on compartment volume rather than shape. Thus, the amount of cytoplasmic material provides a mechanism for regulating the size of intracellular structures.

The mitotic spindle must function in cell types that vary greatly in size, and its dimensions scale with the rapid, reductive cell divisions that accompany early stages of development. The mechanism responsible for this scaling is unclear, because uncoupling cell size from a developmental or cellular context has proven experimentally challenging. We combined microfluidic technology with Xenopus egg extracts to characterize spindle assembly within discrete, geometrically defined volumes of cytoplasm. Reductions in cytoplasmic volume, rather than developmental cues or changes in cell shape, were sufficient to recapitulate spindle scaling observed in Xenopus embryos. Thus, mechanisms extrinsic to the spindle, specifically a limiting pool of cytoplasmic component(s), play a major role in determining spindle size.

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.

Extracellular calcium is crucial for the normal function of muscle spindle sensory afferents. They express multiple calcium buffering proteins. Extracellular calcium is essential for recycling of synaptic‐like vesicles (SLVs) in the terminals and for the stretch‐evoked inward calcium current of the receptor potential. Conversely, removal of calcium from the extracellular medium abolishes stretch‐evoked action potentials (APs). However, the calcium channel(s) involved and mechanism(s) of action are unknown. This study begins identifying the channels involved and their actions. Specific calcium channel toxins, agonists and antagonists were examined for effects on stretch‐evoked muscle spindle afferent discharge, and live spindle sensory terminal labelling with FM1‐43 was used to monitor SLV recycling in adult rat lumbrical muscle. Voltage‐gated calcium channels, particularly P/Q‐type (Cav2.1) and L‐type (Cav1.1–1.4), strongly regulated the firing frequency of APs in response to a standard stretch, probably by regulating the opening of ‘big’, ‘intermediate’ and ‘small’ calcium‐activated potassium channels (KCa), with direct evidence for BK (KCa1.1), SK (most likely KCa2.2) and IK (KCa3.1) involvement. Moreover, calcium from two different sources regulated separate aspects of SLV recycling. Thus, L‐type channel blockers inhibited FM1‐43 release, while TRPV4 (transient receptor potential, vanilloid, type 4) channel blockers entirely inhibited FM1‐43 uptake. No role in SLV recycling was found for P/Q type channels, and no role at all was found for N‐type (Cav2.3) channels. Overall, these studies pinpoint multiple different aspects of calcium signalling, through different channel families, and produce the first evidence of a role for a mechanosensory TRPV4 channel in muscle spindle sensory terminal function. What is the central question of this study? External calcium is essential for muscle spindle stretch‐evoked nerve firing and sensory nerve terminals express multiple calcium‐buffering proteins, yet calcium hardly contributes to stimulus‐evoked potentials: so what is calcium's role? What is the main finding and its importance? Muscle spindles of ex vivo rat muscles revealed multiple roles for calcium. Stretch (TRPV4) and voltage‐activated (L‐type) calcium channels control endo‐ and exocytosis of glutamate, respectively, essential for terminal stretch‐sensitivity. Multiple calcium‐activated potassium channels gated by voltage‐activated (L‐ and P/Q‐type) calcium channels regulate afferent discharge rates encoding muscle length to the CNS.

The stretch reflex is a fundamental component of the motor system that orchestrates the coordinated muscle contractions underlying movement. At the heart of this process lie the muscle spindles (MS), specialized receptors finely attuned to fluctuations in tension within intrafusal muscle fibres. The tension variation in the MS triggers a series of neuronal events including an initial depolarization of sensory type Ia afferents that subsequently causes the activation of motoneurons within the spinal cord 1 , 2 . This neuronal cascade culminates in the execution of muscle contraction, underscoring a presumed closed-loop mechanism between the musculoskeletal and nervous systems. By contrast, here we report the discovery of a new population of macrophages with exclusive molecular and functional signatures within the MS that express the machinery for synthesizing and releasing glutamate. Using mouse intersectional genetics with optogenetics and electrophysiology, we show that activation of MS macrophages (MSMP) drives proprioceptive sensory neuron firing on a millisecond timescale. MSMP activate spinal circuits, motor neurons and muscles by means of a glutamate-dependent mechanism that excites the MS. Furthermore, MSMP respond to neural and muscle activation by increasing the expression of glutaminase, enabling them to convert the uptaken glutamine released by myocytes during muscle contraction into glutamate. Selective silencing or depletion of MSMP in hindlimb muscles disrupted the modulation of the stretch reflex for force generation and sensory feedback correction, impairing locomotor strategies in mice. Our results have identified a new cellular component, the MSMP, that directly regulates neural activity and muscle contraction. The glutamate-mediated signalling of MSMP and their dynamic response to sensory cues introduce a new dimension to our understanding of sensation and motor action, potentially offering innovative therapeutic approaches in conditions that affect sensorimotor function. A population of macrophages with exclusive molecular and functional signatures in the muscle spindles express machinery for synthesizing and releasing glutamate, and a cellular component, the muscle spindle macrophages, directly regulates neural activity and muscle contraction.

Purpose Fibrosis of muscle spindles (sensory organs) in back muscles induced by intervertebral disc (IVD) degeneration could limit transmission of muscle stretch to the sensory receptor and explain the proprioceptive deficits common in back pain. Exercise reduces back muscles fibrosis. This study investigated whether targeted muscle activation via neurostimulation reverses or resolves muscle spindle fibrosis in a model of IVD injury. Methods In eighteen sheep, lumbar (L)1–2 and L3-4 IVD degeneration was induced by partial thickness anulus fibrosis incision and a neurostimulator was implanted. After IVD-degeneration developed for 3 months, neurostimulation of the L2 nerve root activated multifidus in nine randomly selected animals. Multifidus muscle adjacent to the spinous process of L2 (non-stimulated) and L4 (stimulated) was harvested 3 months after activation. Muscle spindles were identified in Van Giessen’s-stained sections. Connective tissue spindle capsule thickness, and cross-sectional area (CSA) of the spindle, its periaxial fluid and sensory elements were measured. Immunofluorescence assays evaluated Collagen-I and -III. Results Multifidus muscle spindle capsule thickness and Collagen-1 were significantly less in the neurostimulation animals than IVD-injury animals at L4 (stimulated muscle) ( P  < 0.05), but not L2 (non-stimulated muscle). Spindle capsule thickness was less in lateral than medial regions. CSA of the muscle spindle and sensory elements was less in neurostimulated animals at L4. Conclusion Targeted multifidus activation reverses or prevents accumulation of connective tissue of the multifidus muscle spindle capsule caused by IVD injury. Reduced fibrosis should maintain sensory function of this important muscle mechanoreceptor and might provide an effective solution to resolve the commonly identified proprioceptive deficits in back pain and maintain healthy spine function.

Muscle spindle proprioceptive receptors play a primary role in encoding the effects of external mechanical perturbations to the body. During externally-imposed stretches of passive, i.e. electrically-quiescent, muscles, the instantaneous firing rates (IFRs) of muscle spindles are associated with characteristics of stretch such as length and velocity. However, even in passive muscle, there are history-dependent transients of muscle spindle firing that are not uniquely related to muscle length and velocity, nor reproduced by current muscle spindle models. These include acceleration-dependent initial bursts, increased dynamic response to stretch velocity if a muscle has been isometric, and rate relaxation, i.e., a decrease in tonic IFR when a muscle is held at a constant length after being stretched. We collected muscle spindle spike trains across a variety of muscle stretch kinematic conditions, including systematic changes in peak length, velocity, and acceleration. We demonstrate that muscle spindle primary afferents in passive muscle fire in direct relationship to muscle force-related variables, rather than length-related variables. Linear combinations of whole muscle-tendon force and the first time derivative of force (dF/dt) predict the entire time course of transient IFRs in muscle spindle Ia afferents during stretch (i.e., lengthening) of passive muscle, including the initial burst, the dynamic response to lengthening, and rate relaxation following lengthening. Similar to acceleration scaling found previously in postural responses to perturbations, initial burst amplitude scaled equally well to initial stretch acceleration or dF/dt, though later transients were only described by dF/dt. The transient increase in dF/dt at the onset of lengthening reflects muscle short-range stiffness due to cross-bridge dynamics. Our work demonstrates a critical role of muscle cross-bridge dynamics in history-dependent muscle spindle IFRs in passive muscle lengthening conditions relevant to the detection and sensorimotor response to mechanical perturbations to the body, and to previously-described history-dependence in perception of limb position.

A functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation. Mitotic spindle assembly is required for proper cell division, but many underlying mechanisms remain unclear. Here, the authors show that NuMa undergoes liquid-liquid phase separation, condensing on spindle poles during mitotic entry and enriching critical components to promote spindle assembly.

In the dividing eukaryotic cell, the spindle assembly checkpoint (SAC) ensures that each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for initiating chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), which inhibits the APC/C and delays chromosome segregation. By cryo-electron microscopy, here we determine the near-atomic resolution structure of a human APC/C–MCC complex (APC/C MCC ). Degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit responsible for substrate interactions. BubR1 also obstructs binding of the initiating E2 enzyme UbcH10 to repress APC/C ubiquitination activity. Conformational variability of the complex enables UbcH10 association, and structural analysis shows how the Cdc20 subunit intrinsic to the MCC (Cdc20 MCC ) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. A high-resolution structure of a complex between the anaphase-promoting complex (APC/C) and the mitotic checkpoint complex (MCC) reveals how MCC interacts with and represses APC/C by obstructing substrate recognition and suppressing E3 ligase activity. Basis of mitotic regulation The spindle assembly checkpoint (SAC) is a surveillance mechanism that detects incorrect chromatid kinetochore attachments and delays chromosome segregation by generating a 'wait anaphase' signal. It is activated via the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex (APC/C), a multimeric E3 ligase. Here, David Barford and colleagues use cryo-electron microscopy to determine near-atomic resolution structures of the APC/C–MCC complex. The structures reveal how MCC interacts with and represses APC/C by obstructing substrate recognition and suppressing E3 ligase activity.

The purpose of meiosis is to generate developmentally competent, haploid gametes with the correct number of chromosomes. For reasons not completely understood, female meiosis is more prone to chromosome segregation errors than meiosis in males, leading to an abnormal number of chromosomes, or aneuploidy, in gametes. Meiotic spindles are the cellular machinery essential for the proper segregation of chromosomes. One unique feature of spindle structures in female meiosis is spindles poles that lack centrioles. The process of building a meiotic spindle without centrioles is complex and requires precise coordination of different structural components, assembly factors, motor proteins, and signaling molecules at specific times and locations to regulate each step. In this review, we discuss the basics of spindle formation during oocyte meiotic maturation focusing on mouse and human studies. Finally, we review different factors that could alter the process of spindle formation and its stability. We conclude with a discussion of how different assisted reproductive technologies could affect spindles and the consequences these perturbations may have for subsequent embryo development. Summary Sentence This review consolidates information about how spindles form in human and mouse oocytes and how this process can be altered. Graphical Abstract