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).

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.

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.

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 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.

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.

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.

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.

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.

Calcium-regulated import of myosin IC into the nucleus.

Myosin IC is a molecular motor involved in intracellular transport, cell motility, and transcription. Its mechanical properties are regulated by calcium via calmodulin binding, and its functions in the nucleus depend on import from the cytoplasm. The import has recently been shown to be mediated by the nuclear localization signal located within the calmodulin-binding domain. In the present paper, it is demonstrated that mutations in the calmodulin-binding sequence shift the intracellular distribution of myosin IC to the nucleus. The redistribution is displayed by isoform B, described originally as the "nuclear myosin," but is particularly pronounced with isoform C, the normally cytoplasmic isoform. Furthermore, experimental elevation of the intracellular calcium concentration induces a rapid import of myosin into the nucleus. The import is blocked by the importin ß inhibitor importazole. These findings are consistent with a mechanism whereby calmodulin binding prevents recognition of the nuclear localization sequence by importin ß, and the steric inhibition of import is released by cell signaling leading to the intracellular calcium elevation. The results establish a mechanistic connection between the calcium regulation of the motor function of myosin IC in the cytoplasm and the induction of its import into the nucleus. © 2016 Wiley Periodicals, Inc.

CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor.

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/ß1 and the components of the LL5ß-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.

Centrobin regulates centrosome function in interphase cells by limiting pericentriolar matrix recruitment.

The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.

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.

An experimental and computational study of effects of microtubule stabilization on T-cell polarity.

T-killer cells eliminate infected and cancerous cells with precision by positioning their centrosome near the interface (immunological synapse) with the target cell. The mechanism of centrosome positioning has remained controversial, in particular the role of microtubule dynamics in it. We re-examined the issue in the experimental model of Jurkat cells presented with a T cell receptor-binding artificial substrate, which permits controlled stimulation and reproducible measurements. Neither 1-microM taxol nor 100-nM nocodazole inhibited the centrosome positioning at the "synapse" with the biomimetic substrate. At the same time, in micromolar taxol but not in nanomolar nocodazole the centrosome adopted a distinct peripheral rather than the normally central position within the synapse. This effect was reproduced in a computational energy-minimization model that assumed no microtubule dynamics, but only a taxol-induced increase in the length of the microtubules. Together, the experimental and computational results indicate that microtubule dynamics are not essential for the centrosome positioning, but that the fit of the microtubule array in the deformed body of the conjugated T cell is a major factor. The possibility of modulating the T-cell centrosome position with well-studied drugs and of predicting their effects in silico appears attractive for designing anti-cancer and antiviral therapies.

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.

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.

A model for mechanotransduction in cardiac muscle: effects of extracellular matrix deformation on autocrine signaling.

We present a computational model and analysis of the dynamic behavior of epidermal growth factor receptor (EGFR) signaling in cardiac muscle tissue, with the aim of exploring transduction of mechanical loading into cellular signaling that could lead to cardiac hypertrophy. For this purpose, we integrated recently introduced models for ligand dynamics within compliant intercellular spaces and for the spatial dynamics of intracellular signaling with a positive feedback autocrine circuit. These kinetic models are here considered in the setting of a tissue consisting of cardiomyocytes and blood capillaries as a structural model for the myocardium. We show that autocrine EGFR signaling can be induced directly by mechanical deformation of the tissue and demonstrate the possibility of self-organization of signaling that is anisotropic on the tissue level and can reflect anisotropy of the mechanical deformation. These predictions point to the potential capabilities of the EGFR autocrine signaling circuit in mechanotransduction and suggest a new perspective on the cardiac hypertrophic response.

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 polarized cell signaling via autocrine circuits: computational model analysis.

Recent studies have suggested that autocrine signaling through epidermal growth factor receptor (EGFR) might be involved in generating or maintaining an intrinsic polarity in tissue cells, possibly via spatial localization of EGFR-mediated signaling. The difficulty of experimental investigation of autocrine signaling makes especially valuable an application of computational modeling for critical hypotheses about the dynamic operation of the underlying signaling circuits, both intracellular and extracellular. Toward this end, we develop and analyze here a spatially distributed dynamic computational model of autocrine EGFR signaling. Under certain conditions, the model spontaneously evolves into a state wherein sustained signaling is spatially localized on smaller than cell dimension, conferring a polarity to the otherwise nonpolar model cell. Conditions of a sufficiently large rate of autocrine EGFR ligand release and of a sufficiently small exogenous ligand concentration are qualitatively consistent with experimental observations of EGFR-mediated migration. Thus, computational analysis supports the concept that autocrine EGFR signaling circuits could play a role in helping generate and/or maintain an intrinsic cell spatial polarity, possibly related to migration as well as tissue organization. We additionally offer particular suggestions for critical nodes in the EGFR signaling circuits governing this self-organization capability.

Centrosome-dependent anisotropic random walk of cytoplasmic vesicles.

We approach the problem of an apparently random movement of small cytoplasmic vesicles and its relationship to centrosome functioning. Motion of small vesicles in the cytoplasm of BSC-1 cells was quantified using computer-assisted microscopy. The vesicles move across the cytoplasm frequently changing their directions with negligible net displacement. The autocorrelation function for consecutive velocities of individual vesicles becomes indistinguishable from zero in 10s. Variance in the displacement is proportional to time. The motion of vesicles is anisotropic: It has diffusivity along the radii drawn from the centrosome several times higher than the tangential diffusivity. This anisotropy is abolished by ultraviolet microbeam irradiation of the centrosome when the microtubule array loses radial structure. We conclude that the motion of the vesicles in the cytoplasm can be described as diffusion-like random walk with centrosome-dependent anisotropy. The present analysis quantitatively corroborates the 'trial and error' model of vesicular transport.