Myosins in the Nucleus.

Although originally characterized as a cytoplasmic protein, myosin of various classes also performs key functions in the nucleus. We review the data concerning the nuclear localization, mechanism of entry, and functional interactions of myosin I, II, V, VI, X, XVI, and XVIII. To date, the first-characterized "nuclear myosin I" (or, in the prevailing nomenclature, myosin IC isoform B) remains the best-studied nuclear myosin, although results are rapidly accumulating that illuminate the roles of other myosin classes, and an outline of a unified picture of myosin functions in the nucleus is beginning to emerge. Reflecting the state of knowledge in this field, the review concentrates on the mechanisms mediating and regulating import of myosin IC into the nucleus and its role, alongside myosin V and VI, in transcription. Myosin functions in chromatin dynamics, epigenetic mechanisms, intranuclear motility, and nuclear export of RNA and protein are also addressed. Partners and regulators of myosin, such as nuclear actin, kinases, and phosphatases are briefly covered. Problem areas are identified and testable hypotheses are offered with an aim of focusing the research efforts on overcoming the gaps on the way toward a systems-level understanding of processes involving nuclear myosins and their place in cell physiology as a whole.

Effect of Palmitic Acid on Exosome-Mediated Secretion and Invasive Motility in Prostate Cancer Cells.

High fat consumption can enhance metastasis and decrease survival in prostate cancer, but the picture remains incomplete on the epidemiological and cell-biological level, impeding progress toward individualized recommendations in the clinic. Recent work has highlighted the role of exosomes secreted by prostate cancer cells in the progression of the disease, particularly in metastatic invasion, and also the utility of targeting these extracellular vesicles for diagnostics, as carriers of disease progression markers. Here, we investigated the question of a potential impact of the chief nutritional saturated fatty acid on the exosome secretion. Palmitic acid decreased the secretion of exosomes in human prostate cancer cells in vitro in a concentration-dependent manner. At the same time, the content of some prospective metastatic markers in the secreted exosomal fraction was also reduced, as was the ability of the cells to invade across extracellular matrix barriers. While by themselves our in vitro results imply that on the cell level, palmitic acid may be beneficial vis-à-vis the course of the disease, they also suggest that, by virtue of the decreased biomarker secretion, palmitic acid has the potential to cause unjustified deprioritization of treatment in obese and lipidemic men.

Calcium and Nuclear Signaling in Prostate Cancer.

Recently, there have been a number of developments in the fields of calcium and nuclear signaling that point to new avenues for a more effective diagnosis and treatment of prostate cancer. An example is the discovery of new classes of molecules involved in calcium-regulated nuclear import and nuclear calcium signaling, from the G protein-coupled receptor (GPCR) and myosin families. This review surveys the new state of the calcium and nuclear signaling fields with the aim of identifying the unifying themes that hold out promise in the context of the problems presented by prostate cancer. Genomic perturbations, kinase cascades, developmental pathways, and channels and transporters are covered, with an emphasis on nuclear transport and functions. Special attention is paid to the molecular mechanisms behind prostate cancer progression to the malignant forms and the unfavorable response to anti-androgen treatment. The survey leads to some new hypotheses that connect heretofore disparate results and may present a translational interest.

CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization.

BACKGROUND: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. METHODS: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. FINDINGS: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. INTERPRETATION: CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Neuronal actin cytoskeleton gain of function in the human brain.

BACKGROUND: While advancements in imaging techniques have led to major strides in deciphering the human brain, successful interventions are elusive and represent some of the most persistent translational gaps in medicine. Human restricted CHRFAM7A has been associated with neuropsychiatric disorders. METHODS: The physiological role of CHRFAM7A in human brain is explored using multiomics approach on 600 post mortem human brain tissue samples. The emerging pathways and mechanistic hypotheses are tested and validated in an isogenic hiPSC model of CHRFAM7A knock-in medial ganglionic eminence progenitors and neurons. FINDINGS: CHRFAM7A is identified as a modulator of intracellular calcium dynamics and an upstream regulator of Rac1. Rac1 activation re-designs the actin cytoskeleton leading to dynamic actin driven remodeling of membrane protrusion and a switch from filopodia to lamellipodia. The reinforced cytoskeleton leads to an advantage to tolerate stiffer mechanical properties of the extracellular environment. INTERPRETATION: CHRFAM7A modifies the actin cytoskeleton to a more dynamic and stiffness resistant state in an α7nAChR dependent manner. CHRFAM7A may facilitate neuronal adaptation to changes in the brain environment in physiological and pathological conditions contributing to risk or recovery. Understanding how CHRFAM7A affects human brain requires human studies in the areas of memory formation and erasure, cognitive reserve, and neuronal plasticity. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti). Also, in part by the International Society for Neurochemistry (ISN) and The Company of Biologists (Nicolas Rosas). ROSMAP is supported by NIA grants P30AG10161, P30AG72975, R01AG15819, R01AG17917. U01AG46152, and U01AG61356.

Rab6 regulates transport and targeting of exocytotic carriers.

Constitutive exocytosis delivers newly synthesized proteins, lipids, and other molecules from the Golgi apparatus to the cell surface. This process is mediated by vesicles, which bud off the trans-Golgi network, move along cytoskeletal filaments, and fuse with the plasma membrane. Here, we show that the small GTPase Rab6 marks exocytotic vesicles and, together with the microtubule plus-end-directed motor kinesin-1, stimulates their processive microtubule-based transport to the cell periphery. Furthermore, Rab6 directs targeting of secretory vesicles to plasma-membrane sites enriched in the cortical protein ELKS, a known Rab6 binding partner. Our data demonstrate that although Rab6 is not essential for secretion, it controls the organization of exocytosis within the cellular space.

Bayesian inference of molecular kinetic parameters from astrocyte calcium imaging data.

Model-based Bayesian inference from high-content data obtained on live specimens is a burgeoning field with demonstrated applications to neuroscience. In parallel, computer vision methods for extracting the calcium signaling information from imaging data have advanced in application to neuronal physiology. Here, we are describing in detail a method we have recently developed to study calcium dynamics in astrocytes, which combines computer vision with model-based Bayesian learning to deduce the most likely molecular kinetic parameters underlying the observed calcium activity. As reported in the companion experimental study, this method allowed us to identify the key molecular changes downstream of a multi-gene deletion modeling the human 22q11.2 deletion syndrome, the most common human microdeletion and the genetic factor with the highest penetrance for schizophrenia.•Methodological details are laid out, from our imaging approach to our adaptation of the VBA-CaBBI algorithm previously developed primarily for brain functional imaging data.•The analytical pipeline is suited for further applications to glial cells and adaptable to other cell types exhibiting complexcalcium dynamics.

Experimental and computational analyses of calcium dynamics in 22q11.2 deletion model astrocytes.

Methods for deriving mechanistic information from intracellular calcium dynamics have largely been applied to neuronal data despite the knowledge of roles of glial cells in behavior, cognition, and psychiatric disorders. Using calcium imaging, computer vision, and Bayesian kinetic inference (BKI), we analyzed calcium dynamics in primary astrocytes derived from control or Df1/+ mice, a model of 22q11.2 deletion (DiGeorge syndrome). Inference of the highest-likelihood molecular kinetic characteristics of intracellular calcium dynamics identified changes in the activity of the sarcoendoplasmic reticulum calcium ATPase (SERCA). Application of a SERCA inhibitor to wild-type astrocytes reproduced the differences detected in Df1/+ astrocytes. Our work reveals the molecular changes driving the calcium kinetics in astrocytes from a 22q11.2 deletion model. BKI can be useful for mechanistically dissecting calcium dynamics in glial cells and formulating and testing hypotheses about underlying molecular mechanisms.

Astrocyte Bioenergetics and Major Psychiatric Disorders.

Ongoing research continues to add new elements to the emerging picture of involvement of astrocyte energy metabolism in the pathophysiology of major psychiatric disorders, including schizophrenia, mood disorders, and addictions. This review outlines what is known about the energy metabolism in astrocytes, the most numerous cell type in the brain, and summarizes the recent work on how specific perturbations of astrocyte bioenergetics may contribute to the neuropsychiatric conditions. The role of astrocyte energy metabolism in mental health and disease is reviewed on the organism, organ, and cell level. Data arising from genomic, metabolomic, in vitro, and neurobehavioral studies is critically analyzed to suggest future directions in research and possible metabolism-focused therapeutic interventions.

Myosin 1C isoform A is a novel candidate diagnostic marker for prostate cancer.

Early diagnosis of prostate cancer is a challenging issue due to the lack of specific markers. Therefore, a sensitive diagnostic marker that is expressed or upregulated exclusively in prostate cancer cells would facilitate diagnostic procedures and ensure a better outcome. We evaluated the expression of myosin 1C isoform A in 5 prostate cell lines, 41 prostate cancer cases, and 11 benign hyperplasias. We analyzed the expression of 12 surface molecules on prostate cancer cells by flow cytometry and analyzed whether high or low myosin 1C isoform A expression could be attributed to a distinct phenotype of prostate cancer cells. Median myosin 1C isoform A expression in prostate cancer samples and cancer cell lines was 2 orders of magnitude higher than in benign prostate hyperplasia. Based on isoform A expression, we could also distinguish clinical stage 2 from clinical stage 3. Among cell lines, PC-3 cells with the highest myosin 1C isoform A level had diminished numbers of CD10/CD13-positive cells and increased numbers of CD29 (integrin ß1), CD38, CD54 (ICAM1) positive cells. The surface phenotype of clinical samples was similar to prostate cancer cell lines with high isoform A expression and could be described as CD10-/CD13- with heterogeneous expression of other markers. Both for cell lines and cancer specimens we observed the strong correlation of high myosin 1C isoform A mRNA expression and elevated levels of CD29 and CD54, suggesting a more adhesive phenotype for cells with high isoform A expression. Compared to normal tissue, prostate cancer samples had also reduced numbers of CD24- and CD38-positive cells. Our data suggest that a high level of myosin 1C isoform A is a specific marker both for prostate cancer cells and prostate cancer cell lines. High expression of isoform A is associated with less activated (CD24/CD38 low) and more adhesive (CD29/CD54 high) surface phenotype compared to benign prostate tissue.

Deterministic mechanical model of T-killer cell polarization reproduces the wandering of aim between simultaneously engaged targets.

T-killer cells of the immune system eliminate virus-infected and tumorous cells through direct cell-cell interactions. Reorientation of the killing apparatus inside the T cell to the T-cell interface with the target cell ensures specificity of the immune response. The killing apparatus can also oscillate next to the cell-cell interface. When two target cells are engaged by the T cell simultaneously, the killing apparatus can oscillate between the two interface areas. This oscillation is one of the most striking examples of cell movements that give the microscopist an unmechanistic impression of the cell's fidgety indecision. We have constructed a three-dimensional, numerical biomechanical model of the molecular-motor-driven microtubule cytoskeleton that positions the killing apparatus. The model demonstrates that the cortical pulling mechanism is indeed capable of orienting the killing apparatus into the functional position under a range of conditions. The model also predicts experimentally testable limitations of this commonly hypothesized mechanism of T-cell polarization. After the reorientation, the numerical solution exhibits complex, multidirectional, multiperiodic, and sustained oscillations in the absence of any external guidance or stochasticity. These computational results demonstrate that the strikingly animate wandering of aim in T-killer cells has a purely mechanical and deterministic explanation.

Mechanotransduction through growth-factor shedding into the extracellular space.

Physical forces elicit biochemical signalling in a diverse array of cells, tissues and organisms, helping to govern fundamental biological processes. Several hypotheses have been advanced that link physical forces to intracellular signalling pathways, but in many cases the molecular mechanisms of mechanotransduction remain elusive. Here we find that compressive stress shrinks the lateral intercellular space surrounding epithelial cells, and triggers cellular signalling via autocrine binding of epidermal growth factor family ligands to the epidermal growth factor receptor. Mathematical analysis predicts that constant rate shedding of autocrine ligands into a collapsing lateral intercellular space leads to increased local ligand concentrations that are sufficient to account for the observed receptor signalling; direct experimental comparison of signalling stimulated by compressive stress versus exogenous soluble ligand supports this prediction. These findings establish a mechanism by which mechanotransduction arises from an autocrine ligand-receptor circuit operating in a dynamically regulated extracellular volume, not requiring induction of force-dependent biochemical processes within the cell or cell membrane.

Self-organization of treadmilling microtubules into a polar array.

The centrosome is normally thought to determine the cell center and to dictate the formation of a radial array of microtubules that defines the spatial organization of cytoplasm. However, experiments indicate the existence of a mechanism for organization of a centered microtubule array that is independent of the centrosome. Here, we formulate a model of treadmilling dynamics of non-centrosomal microtubules that predicts a spontaneously established, polarized distribution of microtubule orientation. Based on this model, we propose that the autonomous ability of non-centrosomal microtubules to form a polarized array arises from their treadmilling within the space constrained by the cell boundary.

Introduction: a practical guide to the systems approach in biology.

This essay provides an informal review of the modern systems-centric biological methodology for the practical researcher. The systems approach is defined, and a generic recipe for employing it in biomedical research is offered. General caveats are discussed that pertain to biological complexity, to explanation in molecular terms, and to bottom-up investigation. An outlook on the development of systems biology is also given.

Effect of polyunsaturated fatty acids on proliferation and survival of prostate cancer cells.

Progression of prostate cancer to lethal forms is marked by emergence of hormone-independent proliferation of the cancer cells. Nutritional and epidemiological studies have indicated that prostate cancer progression is correlated with the consumption of polyunsaturated fatty acids (PUFA). To shed additional light on the cell-level mechanisms of the observed correlation, we compared the sensitivity of hormone-dependent and hormone-independent prostate cancer cells to growth medium supplementation with free PUFAs in a cell proliferation and viability assay. Our data show that the hormone-dependent cells are comparatively insensitive to various PUFAs, at the same time as the growth and viability of hormone-independent cells lines are strongly inhibited by most of the tested PUFAs, whether n-3 or n-6. We speculate that this difference may be at least partially responsible for the observed effects of specific dietary lipids in prostate cancer. The new data strengthen the case for dietary intervention as part of potential new therapeutic strategies seeking to impede prostate cancer progression.

Retractile processes in T lymphocyte orientation on a stimulatory substrate: morphology and dynamics.

T cells of the immune system target infected and tumor cells in crowded tissues with high precision by coming into direct contact with the intended target and orienting the intracellular Golgi apparatus and the associated organelles to the area of the cell-cell contact. The mechanism of this orientation remains largely unknown. To further elucidate it we used three-dimensional microscopy of living T cells presented with an artificial substrate mimicking the target cell surface. The data indicate that long, finger-like processes emanate from the T cell surface next to the intracellular Golgi apparatus. These processes come in contact with the substrate and retract. The retraction accompanies the reorientation of the T cell body which brings the Golgi apparatus closer to the stimulatory substrate. Numerical modeling indicates that considering the forces involved the retraction of a process attached with one end to the cell body near the Golgi apparatus and with the other end to the substrate can bring the Golgi apparatus to the substrate by moving the entire cell body. The dynamic scenarios that are predicted by the quantitative model explain features of the reorientation movements that we measured but could not explain previously. We propose that retraction of the surface processes is a force-generating mechanism contributing to the functional orientation of T lymphocytes.

Fatty Acids and Calcium Regulation in Prostate Cancer.

Prostate cancer is a widespread malignancy characterized by a comparative ease of primary diagnosis and difficulty in choosing the individualized course of treatment. Management of prostate cancer would benefit from a clearer understanding of the molecular mechanisms behind the transition to the lethal, late-stage forms of the disease, which could potentially yield new biomarkers for differential prognosis and treatment prioritization in addition to possible new therapeutic targets. Epidemiological research has uncovered a significant correlation of prostate cancer incidence and progression with the intake (and often co-intake) of fatty acids and calcium. Additionally, there is evidence of the impact of these nutrients on intracellular signaling, including the mechanisms mediated by the calcium ion as a second messenger. The present review surveys the recent literature on the molecular mechanisms associated with the critical steps in the prostate cancer progression, with special attention paid to the regulation of these processes by fatty acids and calcium homeostasis. Testable hypotheses are put forward that integrate some of the recent results in a more unified picture of these phenomena at the interface of cell signaling and metabolism.

Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells.

BACKGROUND: Prostate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated. METHODS: To verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes (GAPDH, YWHAZ, HPRT1) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry. RESULTS: Median normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-). CONCLUSIONS: We report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of MYO 1C A using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens.

Myosin isoform expressed in metastatic prostate cancer stimulates cell invasion.

During metastasis, tumor cells migrate out of their original tissue to invade other organs. Secretion of exosomes and metalloproteases is essential for extracellular matrix remodeling, enabling migration through tissue barriers. Metastatic prostate cancer is differentiated by expression of the rare isoform A of the molecular motor myosin IC, however the function of this isoform remained unknown. Here we show that it contributes causatively to the invasive motility of prostate cancer cells. We found that the isoform associates with metalloprotease-containing exosomes and stimulates their secretion. While the data show that myosin IC is involved in prostate cancer cell migration, migration outside extracellular matrix in vitro proves little affected specifically by isoform A. Nevertheless, this isoform stimulates invasion through extracellular matrix, pointing to a critical role in secretion. Both the secretion and invasion depend on the integrity of the motor and lipid-binding domains of the protein. Our results demonstrate how myosin IC isoform A is likely to function in metastasis, driving secretion of exosomes that enable invasion of prostate cancer cells across extracellular matrix barriers. The new data identify a molecule suitable for a mechanistically grounded development into a marker and target for prognosis, detection, and treatment of invasive prostate cancer.

Contribution of whole-cell optimization via cell body rolling to polarization of T cells.

Directed secretion of cytotoxins or cytokines by T cells during immune response depends on migration of the centrosome in the T cell to the interface with the target cell. The mechanism of the centrosome translocation has been elusive. The presented computational analysis demonstrates that the centrosome should be positioned at the interface if the T cell attempts simultaneously (a) to minimize its surface area, (b) to maximize the interface area, (c) to maintain the cell volume and (d) to straighten the microtubules. Live three-dimensional microscopy and measurements show that the optimal position of the centrosome is achieved in large part (by about 40%) via rolling of the entire T cell body on the target surface; this movement appears to entrain the centrosome. The theoretical and experimental results draw attention to the previously unrecognized role of the whole-cell structure and whole-cell movements in the T cell polarization.