More is different: Reconstituting complexity in microtubule regulation.

Microtubules are dynamic cytoskeletal filaments that undergo stochastic switching between phases of polymerization and depolymerization-a behavior known as dynamic instability. Many important cellular processes, including cell motility, chromosome segregation, and intracellular transport, require complex spatiotemporal regulation of microtubule dynamics. This coordinated regulation is achieved through the interactions of numerous microtubule-associated proteins (MAPs) with microtubule ends and lattices. Here, we review the recent advances in our understanding of microtubule regulation, focusing on results arising from biochemical in vitro reconstitution approaches using purified multiprotein ensembles. We discuss how the combinatory effects of MAPs affect both the dynamics of individual microtubule ends, as well as the stability and turnover of the microtubule lattice. In addition, we highlight new results demonstrating the roles of protein condensates in microtubule regulation. Our overall intent is to showcase how lessons learned from reconstitution approaches help unravel the regulatory mechanisms at play in complex cellular environments.

Collective effects of XMAP215, EB1, CLASP2, and MCAK lead to robust microtubule treadmilling.

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.

CLASPs at a glance.

CLIP-associating proteins (CLASPs) form an evolutionarily conserved family of regulatory factors that control microtubule dynamics and the organization of microtubule networks. The importance of CLASP activity has been appreciated for some time, but until recently our understanding of the underlying molecular mechanisms remained basic. Over the past few years, studies of, for example, migrating cells, neuronal development, and microtubule reorganization in plants, along with in vitro reconstitutions, have provided new insights into the cellular roles and molecular basis of CLASP activity. In this Cell Science at a Glance article and the accompanying poster, we will summarize some of these recent advances, emphasizing how they impact our current understanding of CLASP-mediated microtubule regulation.

CLASPs stabilize the pre-catastrophe intermediate state between microtubule growth and shrinkage.

Cytoplasmic linker-associated proteins (CLASPs) regulate microtubules in fundamental cellular processes. CLASPs stabilize dynamic microtubules by suppressing microtubule catastrophe and promoting rescue, the switch-like transitions between growth and shrinkage. How CLASPs specifically modulate microtubule transitions is not understood. Here, we investigate the effects of CLASPs on the pre-catastrophe intermediate state of microtubule dynamics, employing distinct microtubule substrates to mimic the intermediate state. Surprisingly, we find that CLASP1 promotes the depolymerization of stabilized microtubules in the presence of GTP, but not in the absence of nucleotide. This activity is also observed for CLASP2 family members and a minimal TOG2-domain construct. Conversely, we find that CLASP1 stabilizes unstable microtubules upon tubulin dilution in the presence of GTP. Strikingly, our results reveal that CLASP1 drives microtubule substrates with vastly different inherent stabilities into the same slowly depolymerizing state in a nucleotide-dependent manner. We interpret this state as the pre-catastrophe intermediate state. Therefore, we conclude that CLASPs suppress microtubule catastrophe by stabilizing the intermediate state between growth and shrinkage.

Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe.

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term "stutters." During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.

SSNA1 stabilizes dynamic microtubules and detects microtubule damage.

Sjögren's syndrome nuclear autoantigen-1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells, and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. Our results show that SSNA1 modulates all parameters of microtubule dynamic instability-slowing down the rates of growth, shrinkage, and catastrophe, and promoting rescue. We find that SSNA1 forms stretches along growing microtubule ends and binds cooperatively to the microtubule lattice. Furthermore, SSNA1 is enriched on microtubule damage sites, occurring both naturally, as well as induced by the microtubule severing enzyme spastin. Finally, SSNA1 binding protects microtubules against spastin's severing activity. Taken together, our results demonstrate that SSNA1 is both a potent microtubule-stabilizing protein and a novel sensor of microtubule damage; activities that likely underlie SSNA1's functions on microtubule structures in cells.

Human CLASP2 specifically regulates microtubule catastrophe and rescue.

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins essential for microtubule regulation in many cellular processes. However, the molecular mechanisms underlying CLASP activity are not understood. Here, we use purified protein components and total internal reflection fluorescence microscopy to investigate the effects of human CLASP2 on microtubule dynamics in vitro. We demonstrate that CLASP2 suppresses microtubule catastrophe and promotes rescue without affecting the rates of microtubule growth or shrinkage. Strikingly, when CLASP2 is combined with EB1, a known binding partner, the effects on microtubule dynamics are strongly enhanced. We show that synergy between CLASP2 and EB1 is dependent on a direct interaction, since a truncated EB1 protein that lacks the CLASP2-binding domain does not enhance CLASP2 activity. Further, we find that EB1 targets CLASP2 to microtubules and increases the dwell time of CLASP2 at microtubule tips. Although the temporally averaged microtubule growth rates are unaffected by CLASP2, we find that microtubules grown with CLASP2 display greater variability in growth rates. Our results provide insight into the regulation of microtubule dynamics by CLASP proteins and highlight the importance of the functional interplay between regulatory proteins at dynamic microtubule ends.

Mitochondria localize to the cleavage furrow in mammalian cytokinesis.

Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance.

Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

BACKGROUND: Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported. METHODOLOGY AND PRINCIPAL FINDINGS: To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures. CONCLUSION AND SIGNIFICANCE: Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

A matter of size: Part 2. Evaluating the large-for-gestational-age neonate.

Large for gestational age (LGA) is another designation used to assess and monitor growth throughout the pregnancy and after delivery. Large for gestational age is an abnormal growth descriptor that assists in anticipating neonatal needs pre-and postnatally. Careful monitoring for abnormal growth trends in the fetus is imperative prenatally. The relative size of a neonate affects many aspects of prenatal and postnatal surveillance. Nursing care is guided by the maternal history and the delivery room complications that may occur. Anticipating complications in the delivery room is vital to the survival of LGA neonates. Nursing care for LGA neonates requires knowledge based on these potential complications. A thorough physical assessment with appropriate glucose monitoring and parental education is required. Size matters when it comes to the health and welfare of all sizes of neonates. Anticipatory guidance with prenatal monitoring and education can improve outcomes in the neonate at risk for LGA complications at birth.

Part 1: a matter of size: evaluating the growth-restricted neonate.

The relative size of a neonate impacts many aspects of prenatal and postnatal surveillance and care. The designations of appropriate for gestational age, small-for-gestational age, intrauterine growth restriction, and large-for-gestational age are systematic categorizations used to assess and monitor growth throughout pregnancy and delivery. Each abnormal growth descriptor aids in anticipating neonatal needs after birth because each has the potential for complications related to feeding, glucose utilization, short- and long-term growth, and development. Maternal risk factors that impact the neonate's size-related can have immediate implications in the delivery room as well as significant effects postnatally. Caring for neonates at risk for size complications requires knowledge based on prenatal and postnatal complications. Neonates must be carefully measured and plotted on growth charts to confirm a visual assessment of size. Each growth complication requires individual attention to detail and careful planning to maximize adequate postnatal growth and nutrition. Size matters when it comes to the health and welfare of neonates. Anticipatory guidance can improve outcomes in the neonate at risk for failure to thrive from size complications at birth. Part 1 of this article provides an overview of the size classifications and a discussion of clinical factors that are associated with or contribute to small-for-gestational age births. Once the neonate's size for gestational age is calculated, a focused physical assessment is described along with nursing care and prognostic implications. Part 2 will focus on the physical assessment, nursing care, prognosis, and complications associated with large-for-gestational-age neonates.

The clinical presentation of Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome (EDS), a heterogeneous group of inheritable connective tissue disorders, is attributed to mutations in connective tissue genes. These mutations cause defects in collagen. Collagen, a connective tissue protein that acts like glue, gives strength to the body and provides support and elasticity for movement. Thus, the altered gene affects the mechanical properties of skin, joints, ligaments, and blood vessels. Ehlers-Danlos syndrome is transmitted through autosomal dominant, autosomal recessive, or x-linked patterns of inheritance. The life expectancy of an affected infant varies with the type of EDS. This article provides an overview of the 6 major classifications of EDS, their unique clinical presentations, a focused physical assessment guide, considerations for nursing care, and resources for parents. Ehlers-Danlos syndrome can be a potentially debilitating syndrome. It requires preventative and protective measures starting at birth to preserve joint function to improve infant outcomes. Caring for patients with EDS requires an understanding of the potential associated complications to help minimize the physical and emotional impact of the syndrome and improve the quality of life for affected individuals.