CSPP1 stabilizes microtubules by capping both plus and minus ends.

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting the polymerization. Importantly, CSPP1-bound MTs were resistant to MCAK-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

CENP-I directly targets centromeric DNA to support CENP-A deposition and centromere maintenance.

The enrichment of histone H3 variant CENP-A is the epigenetic mark of centromere and initiates the assembly of the kinetochore at centromere. The kinetochore is a multi-subunit complex that ensures accurate attachment of microtubule centromere and faithful segregation of sister chromatids during mitosis. As a subunit of kinetochore, CENP-I localization at centromere also depends on CENP-A. However, whether and how CENP-I regulates CENP-A deposition and centromere identity remains unclear. Here, we identified that CENP-I directly interacts with the centromeric DNA and preferentially recognizes AT-rich elements of DNA via a consecutive DNA-binding surface formed by conserved charged residues at the end of N-terminal HEAT repeats. The DNA binding-deficient mutants of CENP-I retained the interaction with CENP-H/K and CENP-M, but significantly diminished the centromeric localization of CENP-I and chromosome alignment in mitosis. Moreover, the DNA binding of CENP-I is required for the centromeric loading of newly synthesized CENP-A. CENP-I stabilizes CENP-A nucleosomes upon binding to nucleosomal DNA instead of histones. These findings unveiled the molecular mechanism of how CENP-I promotes and stabilizes CENP-A deposition and would be insightful for understanding the dynamic interplay of centromere and kinetochore during cell cycle.

Phase separation of EB1 guides microtubule plus-end dynamics.

In eukaryotes, end-binding (EB) proteins serve as a hub for orchestrating microtubule dynamics and are essential for cellular dynamics and organelle movements. EB proteins modulate structural transitions at growing microtubule ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. However, the molecular mechanisms and physiochemical properties of the EB1 interaction network remain elusive. Here we show that EB1 formed molecular condensates through liquid-liquid phase separation (LLPS) to constitute the microtubule plus-end machinery. EB1 LLPS is driven by multivalent interactions among different segments, which are modulated by charged residues in the linker region. Phase-separated EB1 provided a compartment for enriching tubulin dimers and other plus-end tracking proteins. Real-time imaging of chromosome segregation in HeLa cells expressing LLPS-deficient EB1 mutants revealed the importance of EB1 LLPS dynamics in mitotic chromosome movements. These findings demonstrate that EB1 forms a distinct physical and biochemical membraneless-organelle via multivalent interactions that guide microtubule dynamics.

SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway.

As an extracellular matrix protein, secreted protein acidic and rich in cysteine (SPARC)-like 1 (SPARCL1) is involved in various cell functions. It was previously implicated in bovine skeletal muscle-derived satellite cell (MDSC) differentiation; however, the underlying mechanism remains unknown. In this study, immunoprecipitation and mass spectrometry revealed that integrin ß1 (ITGB1) combines with SPARCL1. Further, co-immunoprecipitation demonstrated that SPARCL1 interacts with ITGB1. Cell scratch assays explored the influence of SPARCL1 on MDSC migration through ITGB1. In addition, desmin staining for myotube fusion rate and MyoD protein expression results showed that SPARCL1 promotes MDSC early differentiation through ITGB1. Furthermore, Western blotting results demonstrated that SPARCL1 regulates the expression of p-FAK, p-paxillin, vinculin, Cdc42, and Arp2/3 through ITGB1. These findings indicate that SPARCL1 may influence bovine MDSC migration and differentiation through an ITGB1-mediated cell signaling pathway. Herein, we elucidated the mechanism through which SPARCL1 affects MDSC differentiation. Our results provide insight into the molecular mechanism of muscle development and may in the future facilitate skeletal muscle regeneration and treatment.

SPARCL1 promotes C2C12 cell differentiation via BMP7-mediated BMP/TGF-ß cell signaling pathway.

The extracellular matrix (ECM) is known to regulate tissue development and cell morphology, movement, and differentiation. SPARCL1 is an ECM protein, but its role in mouse cell differentiation has not been widely investigated. The results of western blotting and immunofluorescence showed that SPARCL1 is associated with the repair of muscle damage in mice and that SPARCL1 binds to bone morphogenetic protein 7 (BMP7) by regulating BMP/transforming growth factor (TGF)-ß cell signaling. This pathway promotes the differentiation of C2C12 cells. Using CRISPR/Cas9 technology, we also showed that SPARCL1 activates BMP/TGF-ß to promote the differentiation of C2C12 cells. BMP7 molecules were found to interact with SPARCL1 by immunoprecipitation analysis. Western blotting and immunofluorescence were performed to verify the effect of BMP7 on C2C12 cell differentiation. Furthermore, SPARCL1 was shown to influence the expression of BMP7 and activity of the BMP/TGF-ß signaling pathway. Finally, SPARCL1 activation was accompanied by BMP7 inhibition in C2C12 cells, which confirmed that SPARCL1 affects BMP7 expression and can promote C2C12 cell differentiation through the BMP/TGF-ß pathway. The ECM is essential for muscle regeneration and damage repair. This study intends to improve the understanding of the molecular mechanisms of muscle development and provide new treatment ideas for muscle injury diseases.

Thermoresponsive Chiral to Nonchiral Ordering Transformation in the Nematic Liquid-Crystal Phase of Rodlike Viruses: Turning the Survival Strategy of a Virus into Valuable Material Properties.

The current work investigates the thermoresponsive in situ chiral to nonchiral ordering transformation of a rodlike virus in the naturally assembled state-the chiral nematic liquid crystal (CLC) phase. We take this as an elegant example of reconfigurable self-assembly, through which it is possible to realize in situ transformation from one assembled state to another without disrupting the preformed assembly in general or going through a secondary assembling procedure of the disassembled building blocks. The detailed investigation presented here reveals many unique characteristics of the thermoresponsive 3D chiral ordering of rodlike viruses induced by heat stress. The chiral to nonchiral ordering transformation is highly reversible in the temperature range of up to 60 °C and can be repeated many times. There exists a critical temperature around 40 °C which is independent of the ionic strength and virus concentration. Such reconfigurable ordering in the CLC phase stems from the intrinsic structure change of constituent coat proteins without disrupting the structural integrity of the virus, as revealed by three analytical techniques targeting levels ranging from the molecular, secondary conformation of the constituent proteins to the whole single virus, respectively. Such structural flexibility, also termed polymorphism, is relative to the survival strategies of a biological organism such as the virus and can be transformed into very precious material properties. The potential of the virus-based CLC phase as the chiral matrix to regulate chiro-optical properties of gold nanorods is also presented.

Stimuli responsive chiral liquid crystal phases of phenylboronic acid functionalized rodlike viruses and their interaction with biologically important diols.

The rodlike M13 viruses with chemically decorated phenylboronic acid moieties form pH responsive chiral nematic liquid crystal (LC) phases. Binding with biologically important diols results in LC phases with microstructures that closely correlate with the molecular structure of the diols and can be conveniently discerned by visual cues.