Time-varying mobility and turnover of actomyosin ring components during cytokinesis in Schizosaccharomyces pombe.

Cytokinesis in many eukaryotes is dependent on a contractile actomyosin ring (AMR), composed of F-actin, myosin II, and other actin and myosin II regulators. Through fluorescence recovery after photobleaching experiments, many components of the AMR have been shown to be mobile and to undergo constant exchange with the cytosolic pools. However, how the mobility of its components changes at distinct stages of mitosis and cytokinesis has not been addressed. Here, we describe the mobility of eight Schizosaccharomyces pombe AMR proteins at different stages of mitosis and cytokinesis using an approach we have developed. We identified three classes of proteins, which showed 1) high (Ain1, Myo2, Myo51), 2) low (Rng2, Mid1, Myp2, Cdc12), and 3) cell cycle-dependent (Cdc15) mobile fractions. We observed that the F-BAR protein Cdc15 undergoes a 20-30% reduction in its mobile fraction after spindle breakdown and initiation of AMR contraction. Moreover, our data indicate that this change in Cdc15 mobility is dependent on the septation initiation network (SIN). Our work offers a novel strategy for estimating cell cycle-dependent mobile protein fractions in cellular structures and provides a valuable dataset, that is of interest to researchers working on cytokinesis.

Importin binding mediates the intramolecular regulation of anillin during cytokinesis.

Cytokinesis occurs by the ingression of an actomyosin ring that cleaves a cell into two daughters. This process is tightly controlled to avoid aneuploidy, and we previously showed that active Ran coordinates ring positioning with chromatin. Active Ran is high around chromatin, and forms an inverse gradient to cargo-bound importins. We found that the ring component anillin contains a nuclear localization signal (NLS) that binds to importin and is required for its function during cytokinesis. Here we reveal the mechanism whereby importin binding favors a conformation required for anillin's recruitment to the equatorial cortex. Active RhoA binds to the RhoA-binding domain causing an increase in accessibility of the nearby C2 domain containing the NLS. Importin binding subsequently stabilizes a conformation that favors interactions for cortical recruitment. In addition to revealing a novel mechanism for the importin-mediated regulation of a cortical protein, we also show how importin binding positively regulates protein function.

Stromal fibroblast-derived MFAP5 promotes the invasion and migration of breast cancer cells via Notch1/slug signaling.

BACKGROUND: The tumor microenvironment (TME) regulates tumor progression, and cancer-associated fibroblasts (CAFs) are the primary stromal components of the TME, with the potential to drive tumor metastasis via the secretion of paracrine factors, but the specific mechanisms driving this process have not been defined. METHODS: Proteins secreted from CAFs and normal fibroblasts (NFs) were analyzed via proteomic analysis (fold change > 2, p < 0.05) to identify tumor-promoting proteins secreted by CAFs. RESULTS: Proteomic analysis revealed that microfibrillar-associated protein 5 (MFAP5) is preferentially expressed and secreted by CAFs relative to NFs, which was confirmed by Western blotting and RT-qPCR. Transwell and wound healing assays confirmed that MFAP5 is secreted by CAFs, and drives the invasion and migration of MCF7 breast cancer cells. We further found that in MCF7 cells MFAP5 promoted epithelial-mesenchymal transition, activating Notch1 signaling and consequently upregulating NICD1 and slug. When Notch1 was knocked down in MCF7 cells, the ability of MFAP5 to promote invasion and migration decreased. CONCLUSION: CAFs promote cancer cells invasion and migration via MFAP5 secretion and activation of the Notch1/slug signaling. These data highlight this pathway as a therapeutic target to disrupt tumor progression through the interference of CAF-tumor crosstalk.

Automated segmentation of cardiomyocyte Z-disks from high-throughput scanning electron microscopy data.

BACKGROUND: With the advent of new high-throughput electron microscopy techniques such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion-beam scanning electron microscopy (FIB-SEM) biomedical scientists can study sub-cellular structural mechanisms of heart disease at high resolution and high volume. Among several key components that determine healthy contractile function in cardiomyocytes are Z-disks or Z-lines, which are located at the lateral borders of the sarcomere, the fundamental unit of striated muscle. Z-disks play the important role of anchoring contractile proteins within the cell that make the heartbeat. Changes to their organization can affect the force with which the cardiomyocyte contracts and may also affect signaling pathways that regulate cardiomyocyte health and function. Compared to other components in the cell, such as mitochondria, Z-disks appear as very thin linear structures in microscopy data with limited difference in contrast to the remaining components of the cell. METHODS: In this paper, we propose to generate a 3D model of Z-disks within single adult cardiac cells from an automated segmentation of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The proposed fully automated segmentation scheme is comprised of three main modules including "pre-processing", "segmentation" and "refinement". We represent a simple, yet effective model to perform segmentation and refinement steps. Contrast stretching, and Gaussian kernels are used to pre-process the dataset, and well-known "Sobel operators" are used in the segmentation module. RESULTS: We have validated our model by comparing segmentation results with ground-truth annotated Z-disks in terms of pixel-wise accuracy. The results show that our model correctly detects Z-disks with 90.56% accuracy. We also compare and contrast the accuracy of the proposed algorithm in segmenting a FIB-SEM dataset against the accuracy of segmentations from a machine learning program called Ilastik and discuss the advantages and disadvantages that these two approaches have. CONCLUSIONS: Our validation results demonstrate the robustness and reliability of our algorithm and model both in terms of validation metrics and in terms of a comparison with a 3D visualisation of Z-disks obtained using immunofluorescence based confocal imaging.

Matrigel patterning reflects multicellular contractility.

Non-muscle myosin II (NMII)-induced multicellular contractility is essential for development, maintenance and remodeling of tissue morphologies. Dysregulation of the cytoskeleton can lead to birth defects or enable cancer progression. We demonstrate that the Matrigel patterning assay, widely used to characterize endothelial cells, is a highly sensitive tool to evaluate cell contractility within a soft extracellular matrix (ECM) environment. We propose a computational model to explore how cell-exerted contractile forces can tear up the cell-Matrigel composite material and gradually remodel it into a network structure. We identify measures that are characteristic for cellular contractility and can be obtained from image analysis of the recorded patterning process. The assay was calibrated by inhibition of NMII activity in A431 epithelial carcinoma cells either directly with blebbistatin or indirectly with Y27632 Rho kinase inhibitor. Using Matrigel patterning as a bioassay, we provide the first functional demonstration that overexpression of S100A4, a calcium-binding protein that is frequently overexpressed in metastatic tumors and inhibits NMIIA activity by inducing filament disassembly, effectively reduces cell contractility.

Functional analyses of an axonemal inner-arm dynein complex in the bloodstream form of Trypanosoma brucei uncover its essential role in cytokinesis initiation.

The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility-generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner-arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner-arm dynein heavy chain TbIAD5-1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5-1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5-1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner-arm dynein complex in cell division, and provided molecular insights into the flagellum motility-mediated cytokinesis initiation in the bloodstream form of T. brucei.

An essential contractile ring protein controls cell division in Plasmodium falciparum.

During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.

Identification and Characterization of the Anillin Gene in Silkworm.

Anillin is an actin binding protein and plays crucial roles during mitotic cell cycle progression in metazoan. However, the sequence and functions of the Anillin gene have not been yet characterized in the silkworm, Bombyx mori. In this study, we cloned the full-length cDNA sequence of the silkworm Anillin (BmAnillin) gene. The deduced amino acid sequence for BmAnillin protein comprises an Anillin homology region (AHR) covering an Anillin homology domain and a pleckstrin homology domain. Phylogenetic analysis and multiple alignments of the Anillin genes from silkworm and other organisms indicated evolutionary conservation in the AHR containing conserved phosphorylation sites. Reverse transcription-PCR experiments confirmed that the BmAnillin gene was highly expressed during larval development of gonads in which cells undergo mitotic cycles and exhibited an unexpected high expression in silk gland with endocycle during larval molting. RNA interference-mediated knockdown of the BmAnillin gene in silkworm BmN4-SID1 cells derived from ovary disrupted chromosome separation and resulted in a loss of the F-actin filament at cleavage furrow during anaphase, suggesting that the BmAnillin gene is essential for cytokinesis in silkworm.

Anillin facilitates septin assembly to prevent pathological outfoldings of central nervous system myelin.

Myelin serves as an axonal insulator that facilitates rapid nerve conduction along axons. By transmission electron microscopy, a healthy myelin sheath comprises compacted membrane layers spiraling around the cross-sectioned axon. Previously we identified the assembly of septin filaments in the innermost non-compacted myelin layer as one of the latest steps of myelin maturation in the central nervous system (CNS) (Patzig et al., 2016). Here we show that loss of the cytoskeletal adaptor protein anillin (ANLN) from oligodendrocytes disrupts myelin septin assembly, thereby causing the emergence of pathological myelin outfoldings. Since myelin outfoldings are a poorly understood hallmark of myelin disease and brain aging we assessed axon/myelin-units in Anln-mutant mice by focused ion beam-scanning electron microscopy (FIB-SEM); myelin outfoldings were three-dimensionally reconstructed as large sheets of multiple compact membrane layers. We suggest that anillin-dependent assembly of septin filaments scaffolds mature myelin sheaths, facilitating rapid nerve conduction in the healthy CNS.

Regulation and Assembly of Actomyosin Contractile Rings in Cytokinesis and Cell Repair.

Cytokinesis and single-cell wound repair both involve contractile assemblies of filamentous actin (F-actin) and myosin II organized into characteristic ring-like arrays. The assembly of these actomyosin contractile rings (CRs) is specified spatially and temporally by small Rho GTPases, which trigger local actin polymerization and myosin II contractility via a variety of downstream effectors. We now have a much clearer view of the Rho GTPase signaling cascade that leads to the formation of CRs, but some factors involved in CR positioning, assembly, and function remain poorly understood. Recent studies show that this regulation is multifactorial and goes beyond the long-established Ca2+ -dependent processes. There is substantial evidence that the Ca2+ -independent changes in cell shape, tension, and plasma membrane composition that characterize cytokinesis and single-cell wound repair also regulate CR formation. Elucidating the regulation and mechanistic properties of CRs is important to our understanding of basic cell biology and holds potential for therapeutic applications in human disease. In this review, we present a primer on the factors influencing and regulating CR positioning, assembly, and contraction as they occur in a variety of cytokinetic and single-cell wound repair models. Anat Rec, 301:2051-2066, 2018. © 2018 Wiley Periodicals, Inc.

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma.

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies.

Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force-dependence.

Focal adhesions (FAs) are subcellular regions at the micrometer scale that link the cell to the surrounding microenvironment and control vital cell functions. However, the spatial architecture of FAs remains unclear at the nanometer scale. We used two-color and three-color super-resolution stimulated emission depletion microscopy to determine the spatial distributions and co-localization of endogenous FA components in fibroblasts. Our data indicate that adhesion proteins inside, but not outside, FAs are organized into nanometer size units of multi-protein assemblies. The loss of contractile force reduced the nanoscale co-localization between different types of proteins, while it increased this co-localization between markers of the same type. This suggests that actomyosin-dependent force exerts a nonrandom, specific, control of the localization of adhesion proteins within cell-matrix adhesions. These observations are consistent with the possibility that proteins in cell-matrix adhesions are assembled in nanoscale particles, and that force regulates the localization of the proteins therein in a protein-specific manner. This detailed knowledge of how the organization of FA components at the nanometer scale is linked to the capacity of the cells to generate contractile forces expands our understanding of cell adhesion in health and disease.

Diversification of the muscle proteome through alternative splicing.

BACKGROUND: Skeletal muscles express a highly specialized proteome that allows the metabolism of energy sources to mediate myofiber contraction. This muscle-specific proteome is partially derived through the muscle-specific transcription of a subset of genes. Surprisingly, RNA sequencing technologies have also revealed a significant role for muscle-specific alternative splicing in generating protein isoforms that give specialized function to the muscle proteome. MAIN BODY: In this review, we discuss the current knowledge with respect to the mechanisms that allow pre-mRNA transcripts to undergo muscle-specific alternative splicing while identifying some of the key trans-acting splicing factors essential to the process. The importance of specific splicing events to specialized muscle function is presented along with examples in which dysregulated splicing contributes to myopathies. Though there is now an appreciation that alternative splicing is a major contributor to proteome diversification, the emergence of improved "targeted" proteomic methodologies for detection of specific protein isoforms will soon allow us to better appreciate the extent to which alternative splicing modifies the activity of proteins (and their ability to interact with other proteins) in the skeletal muscle. In addition, we highlight a continued need to better explore the signaling pathways that contribute to the temporal control of trans-acting splicing factor activity to ensure specific protein isoforms are expressed in the proper cellular context. CONCLUSIONS: An understanding of the signal-dependent and signal-independent events driving muscle-specific alternative splicing has the potential to provide us with novel therapeutic strategies to treat different myopathies.

Why we need mechanics to understand animal regeneration.

Mechanical forces are an important contributor to cell fate specification and cell migration during embryonic development in animals. Similarities between embryogenesis and regeneration, particularly with regards to pattern formation and large-scale tissue movements, suggest similarly important roles for physical forces during regeneration. While the influence of the mechanical environment on stem cell differentiation in vitro is being actively exploited in the fields of tissue engineering and regenerative medicine, comparatively little is known about the role of stresses and strains acting during animal regeneration. In this review, we summarize published work on the role of physical principles and mechanical forces in animal regeneration. Novel experimental techniques aimed at addressing the role of mechanics in embryogenesis have greatly enhanced our understanding at scales from the subcellular to the macroscopic - we believe the time is ripe for the field of regeneration to similarly leverage the tools of the mechanobiological research community.

Actomyosin contractility regulators stabilize the cytoplasmic bridge between the two primordial germ cells during Caenorhabditis elegans embryogenesis.

Stable cytoplasmic bridges arise from failed cytokinesis, the last step of cell division, and are a key feature of syncytial architectures in the germline of most metazoans. Whereas the Caenorhabditiselegans germline is syncytial, its formation remains poorly understood. We found that the germline precursor blastomere, P4 , fails cytokinesis, leaving a stable cytoplasmic bridge between the two daughter cells, Z2 and Z3 Depletion of several regulators of actomyosin contractility resulted in a regression of the membrane partition between Z2 and Z3, indicating that they are required to stabilize the cytoplasmic bridge. Epistatic analysis revealed a pathway in which Rho regulators promote accumulation of the noncannonical anillin ANI-2 at the stable cytoplasmic bridge, which in turns promotes the accumulation of the nonmuscle myosin II NMY-2 and the midbody component CYK-7 at the bridge, in part by limiting the accumulation of canonical anillin ANI-1. Our results uncover key steps in C. elegans germline formation and define a set of conserved regulators that are enriched at the primordial germ cell cytoplasmic bridge to ensure its stability during embryonic development.

Critical Illness Myopathy (CIM) and Ventilator-Induced Diaphragm Muscle Dysfunction (VIDD): Acquired Myopathies Affecting Contractile Proteins.

Critical care and intensive care units (ICUs) have undergone dramatic changes and improvements in recent years, and critical care is today one of the fastest growing hospital disciplines. Significant improvements in treatments, removal of inefficient and harmful interventions, and introduction of advanced technological support systems have improved survival among critically ill ICU patients. However, the improved survival is associated with an increased number of patients with complications related to modern critical care. Severe muscle wasting and impaired muscle function are frequently observed in immobilized and mechanically ventilated ICU patients. Approximately 30% of mechanically ventilated and immobilized ICU patients for durations of five days and longer develop generalized muscle paralysis of all limb and trunk muscles. These patients typically have intact sensory and cognitive functions, a condition known as critical illness myopathy (CIM). Mechanical ventilation is a lifesaving treatment in critically ill ICU patients; however, the being on a ventilator creates dependence, and the weaning process occupies as much as 40% of the total time of mechanical ventilation. Furthermore, 20% to 30% of patients require prolonged intensive care due to ventilator-induced diaphragm dysfunction (VIDD), resulting in poorer outcomes, and greatly increased costs to health care providers. Our understanding of the mechanisms underlying both CIM and VIDD has increased significantly in the past decade and intervention strategies are presently being evaluated in different experimental models. This short review is restricted CIM and VIDD pathophysiology rather than giving a comprehensive review of all acquired muscle wasting conditions associated with modern critical care. © 2017 American Physiological Society. Compr Physiol 7:105-112, 2017.

The microfibril-associated glycoproteins (MAGPs) and the microfibrillar niche.

The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFß family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvß3 integrins via a RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish resulted in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFß signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation.