Small organelle, big responsibility: the role of centrosomes in development and disease

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development.

Download Full-text

Sphingosine 1-phosphate in inflammation

Journal of Clinical and Nursing Research ◽

10.26689/jcnr.v4i6.1610 ◽

2020 ◽

Vol 4 (6) ◽

Author(s):

Lijuan Li ◽

Lixia An ◽

Lifang Li ◽

Yongjuan Zhao

Keyword(s):

Plasma Membrane ◽

Drug Targets ◽

Therapeutic Potential ◽

Cell Types ◽

In Vivo Models ◽

Integral Role ◽

Sphingosine 1 Phosphate ◽

And Migration

Sphingolipids are formed via the metabolism of sphingomyelin, aconstituent of the plasma membrane, or by denovosynthesis. Enzymatic pathways result in the formation of several different lipid mediators, which are known to have important roles in many cellular processes, including proliferation, apoptosis and migration. Several studies now suggest that these sphingolipid mediators, including ceramide, ceramide 1-phosphate and sphingosine 1-phosphate (S1P), are likely to have an integral role in in?ammation. This can involve, for example, activation of pro-in?ammatory transcription factors in different cell types and induction of cyclooxygenase-2, leading to production of pro-in?ammatory prostaglandins. The mode of action of each sphingolipid is different. Increased ceramide production leads to the formation of ceramide-rich areas of the membrane, which may assemble signalling complexes, whereas S1P acts via high-af?nity G-protein-coupled S1P receptors on the plasma membrane. Recent studies have demonstrated that in vitro effects of sphingolipids on in?ammation can translate into in vivo models. This review will highlight the areas of research where sphingolipids are involved in in?ammation and the mechanisms of action of each mediator. In addition, the therapeutic potential of drugs that alter sphingolipid actions will be examined with reference to disease states, such as asthma and in?ammatory bowel disease, which involve important in?ammatory components. A signi?cant body of research now indicates that sphingolipids are intimately involved in the in?ammatory process and recent studies have demonstrated that these lipids, together with associated enzymes and receptors, can provide effective drug targets for the treatment of pathological in?ammation.

Download Full-text

Mitogen Kinase Kinase (MKK7) controls cytokine production in vitro and in vivo in mice

10.1101/2021.06.25.449967 ◽

2021 ◽

Author(s):

Amada D. Caliz ◽

Hyung-Jin Yoo ◽

Anastassiia Vertii ◽

Cathy Tournier ◽

Roger J. Davis ◽

...

Keyword(s):

Cytokine Production ◽

Cell Types ◽

Minor Role ◽

Cellular Processes ◽

Mitogen Activated Protein ◽

And Migration ◽

Jnk Activation

Mitogen kinase kinase 4 (MKK4) and Mitogen kinase kinase 7 (MKK7) are members of the MAP2K family which can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both, c-Jun N-terminal Kinase (JNK) and p38 MAPK, whereas MKK7 only activates JNK in response to different stimuli. The stimuli as well as cell type determine the choice of MAP2K member that mediates the response. In a variety of cell types, the MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK7 and MKK4 contributes to innate immune response in macrophages as well as during inflammation in vivo. To address this question and elucidate the role of MKK7 and MKK4 in macrophage and in vivo, we developed MKK7- and MKK4-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for LPS induced cytokine production and migration which appears to be a major contributor to the inflammatory response in vivo. Whereas MKK4 plays a significant but minor role in cytokine production in vivo.

Download Full-text

The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts

10.21203/rs.2.18103/v4 ◽

2020 ◽

Author(s):

Nisha Venugopal ◽

Ananga Ghosh ◽

Hardik Gala ◽

Ajoy Aloysius ◽

Neha Vyas ◽

...

Keyword(s):

Cell Cycle ◽

Primary Cilium ◽

Developmental Disorders ◽

Adult Stem Cells ◽

Primary Cilia ◽

Translational Repressor ◽

Elevated Protein ◽

Proliferative Signaling

Abstract Background Reversible cell cycle arrest (quiescence/G0) is characteristic of adult stem cells and is actively controlled at multiple levels. Quiescent cells also extend a primary cilium, which functions as a signaling hub. Primary cilia have been shown to be important in multiple developmental processes, and are implicated in numerous developmental disorders. Although the association of the cilium with G0 is established, the role of the cilium in the control of the quiescence program is still poorly understood. Results Primary cilia are dynamically regulated across different states of cell cycle exit in skeletal muscle myoblasts: quiescent myoblasts elaborate a primary cilium in vivo and in vitro , but terminally differentiated myofibers do not. Myoblasts where ciliogenesis is ablated using RNAi against a key ciliary assembly protein (IFT88) can exit the cell cycle but display an altered quiescence program and impaired self-renewal. Specifically, the G0 transcriptome in IFT88 knockdown cells is aberrantly enriched for G2/M regulators, suggesting a focused repression of this network by the cilium. Cilium-ablated cells also exhibit features of activation including enhanced activity of Wnt and mitogen signaling and elevated protein synthesis via inactivation of the translational repressor 4E-BP1. Conclusions Taken together, our results show that the primary cilium integrates and dampens proliferative signaling, represses translation and G2/M genes, and is integral to the establishment of the quiescence program.

Download Full-text

HIV-1 Tat and heparan sulfate proteoglycan interaction: a novel mechanism of lymphocyte adhesion and migration across the endothelium

Blood ◽

10.1182/blood-2009-01-198945 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3335-3342 ◽

Cited By ~ 33

Author(s):

Chiara Urbinati ◽

Stefania Nicoli ◽

Mauro Giacca ◽

Guido David ◽

Simona Fiorentini ◽

...

Keyword(s):

Heparan Sulfate ◽

Cell Types ◽

Lymphoid Cells ◽

Blood Monocytes ◽

Lymphocyte Adhesion ◽

And Migration ◽

B Lymphoid ◽

Hiv 1

Abstract The HIV-1 transactivating factor Tat accumulates on the surface of endothelium by interacting with heparan sulfate proteoglycans (HSPGs). Tat also interacts with B-lymphoid Namalwa cells but only when these overexpress HSPGs after syndecan-1 cDNA transfection (SYN-NCs). Accordingly, SYN-NCs, but not mock-transfected cells, adhere to endothelial cells (ECs) when Tat is bound to the surface of either one of the 2 cell types or when SYN-NCs are transfected with a Tat cDNA. Moreover, endogenously produced Tat bound to cell-surface HSPGs mediates cell adhesion of HIV+ ACH-2 lymphocytes to the endothelium. This heterotypic lymphocyte-EC interaction is prevented by HSPG antagonist or heparinase treatment, but not by integrin antagonists and requires the homodimerization of Tat protein. Tat tethered to the surface of SYN-NCs or of peripheral blood monocytes from healthy donors promotes their transendothelial migration in vitro in response to CXCL12 or CCL5, respectively, and SYN-NC extravasation in vivo in a zebrafish embryo model of inflammation. In conclusion, Tat homodimers bind simultaneously to HSPGs expressed on lymphoid and EC surfaces, leading to HSPG/Tat-Tat/HSPG quaternary complexes that physically link HSPG-bearing lymphoid cells to the endothelium, promoting their extravasation. These data provide new insights about how lymphoid cells extravasate during HIV infection.

Download Full-text

The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts

10.21203/rs.2.18103/v1 ◽

2019 ◽

Author(s):

Nisha Venugopal ◽

Ananga Ghosh ◽

Hardik Gala ◽

Ajoy Aloysius ◽

Neha Vyas ◽

...

Keyword(s):

Cell Cycle ◽

Primary Cilium ◽

Developmental Disorders ◽

Adult Stem Cells ◽

Primary Cilia ◽

Translational Repressor ◽

Elevated Protein ◽

Proliferative Signaling

Abstract Background: Reversible cell cycle arrest (quiescence/G0) is characteristic of adult stem cells and is actively controlled at multiple levels. Quiescent cells also extend a primary cilium, which functions as a signaling hub. Primary cilia have been shown to be important in multiple developmental processes, and are implicated in numerous developmental disorders. Although the association of the cilium with G0 is established, the role of the cilium in the control of the quiescence program is still poorly understood.Results: Primary cilia are dynamically regulated across different states of cell cycle exit in skeletal muscle myoblasts: quiescent myoblasts elaborate a primary cilium in vivo and in vitro , but terminally differentiated myofibers do not. Myoblasts where ciliogenesis is ablated using RNAi against a key ciliary assembly protein (IFT88) can exit the cell cycle but display an altered quiescence program and impaired self-renewal. Specifically, the G0 transcriptome in IFT88 knockdown cells is aberrantly enriched for G2/M regulators, suggesting a focused repression of this network by the cilium. Cilium-ablated cells also exhibit features of activation including enhanced activity of Wnt and mitogen signaling and elevated protein synthesis via inactivation of the translational repressor 4E-BP1.Conclusions: Taken together, our results show that the primary cilium integrates and dampens proliferative signaling, represses translation and G2/M genes, and is integral to the establishment of the quiescence program.

Download Full-text

Artificial Intelligence Approaches to Assessing Primary Cilia

10.1101/2021.02.03.429602 ◽

2021 ◽

Author(s):

Ruchi Bansal ◽

Staci E. Engle ◽

Tisianna K. Kamba ◽

Kathryn Brewer ◽

Wesley R. Lewis ◽

...

Keyword(s):

Artificial Intelligence ◽

Primary Cilia ◽

Cell Types ◽

Staining Intensity ◽

Brain Regions ◽

Data Sets ◽

Treatment Groups ◽

Neuronal Cilia

ABSTRACTCilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize neuronal cilia in images from both in vivo and in vitro samples. After using our trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and colocalization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.SUMMARYThe use of Artificial Intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

Download Full-text

Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids

10.1101/2021.02.18.431674 ◽

2021 ◽

Author(s):

Dilara Sen ◽

Alexis Voulgaropoulos ◽

Albert J. Keung

Keyword(s):

Stem Cell Differentiation ◽

Cell Types ◽

Brain Regions ◽

Embryoid Bodies ◽

Model Systems ◽

Human Model ◽

Spatial Factors ◽

Neural Lineage

ABSTRACTBackgroundBiophysical factors such as shape and mechanical forces are known to play crucial roles in stem cell differentiation, embryogenesis and neurodevelopment. However, the complexity and experimental challenges capturing such early stages of development, and ethical concerns associated with human embryo and fetal research, limit our understanding of how these factors affect human brain organogenesis. Human cerebral organoids (hCO) are attractive models due to their ability to model important brain regions and transcriptomics of early in vivo brain development. Furthermore, they provide three-dimensional environments that better mimic the in vivo environment. To date, they have been used to understand the effects of genetics and soluble factors on neurodevelopment. Establishing links between spatial factors and hCO development will require the development of new approaches.ResultsHere, we investigated the effects of early geometric confinements on transcriptomic changes during hCO differentiation. Using a custom and tunable agarose microwell platform we generated embryoid bodies (EB) of diverse shapes and then further differentiated those EBs to whole brain hCOs. Our results showed that the microwells did not have negative gross impacts on the ability of the hCOs to differentiate generally towards neural fates, and there were clear shape dependent effects on neural lineage specification. In particular, we observed that non-spherical shapes showed signs of altered neurodevelopmental kinetics and favored the development of medial ganglionic eminence-associated brain regions and cell types over cortical regions.ConclusionsThe findings presented here suggest a role for spatial factors in brain region specification during hCO development. Understanding these spatial patterning factors will not only improve understanding of in vivo development and differentiation, but also provide important handles with which to advance and improve control over human model systems for in vitro applications.

Download Full-text

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22179364 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9364

Author(s):

Amada D. Caliz ◽

Hyung-Jin Yoo ◽

Anastassiia Vertii ◽

Ana C. Dolan ◽

Cathy Tournier ◽

...

Keyword(s):

Cytokine Production ◽

Cell Types ◽

Minor Role ◽

Cellular Processes ◽

Mitogen Activated Protein ◽

And Migration ◽

Jnk Activation

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.

Download Full-text

Simvastatin mediates inhibition of exosome synthesis, localization and secretion via multicomponent interventions

Scientific Reports ◽

10.1038/s41598-019-52765-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Ankur Kulshreshtha ◽

Swati Singh ◽

Mohd Ahmad ◽

Kritika Khanna ◽

Tanveer Ahmad ◽

...

Keyword(s):

Important Implication ◽

Cell Types ◽

Model Systems ◽

Biological Processes ◽

Exosome Biogenesis ◽

Health And Disease ◽

Multicomponent Interventions ◽

Cholesterol Lowering Effect

Abstract Discovery of exosomes as modulator of cellular communication has added a new dimension to our understanding of biological processes. Exosomes influence the biological systems by mediating trans-communication across tissues and cells, which has important implication for health and disease. In absence of well-characterized modulators of exosome biogenesis, an alternative option is to target pathways generating important exosomal components. Cholesterol represents one such essential component required for exosomal biogenesis. We initiated this study to test the hypothesis that owing to its cholesterol lowering effect, simvastatin, a HMG CoA inhibitor, might be able to alter exosome formation and secretion. Simvastatin was tested for its effect on exosome secretion under various in-vitro and in-vivo settings and was found to reduce the secretion of exosome from various cell-types. It was also found to alter the levels of various proteins important for exosome production. Murine model of Acute Airway Inflammation was used for further validation of our findings. We believe that the knowledge acquired in this study holds potential for extension to other exosome dominated pathologies and model systems.

Download Full-text

Abstract 187: c-Kit Biology Revealed by Two Transgenic Reporter Models.

Circulation Research ◽

10.1161/res.121.suppl_1.187 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Natalie A Gude ◽

Fareheh Firouzi ◽

Kristine Nguyen ◽

Christina Payne ◽

Veronica Sacchi ◽

...

Keyword(s):

Flow Cytometry ◽

Transgenic Mouse ◽

Cardiac Myocyte ◽

Biological Significance ◽

Cell Types ◽

Experimental Models ◽

Model Systems ◽

Transgenic Lines

Background: The biological significance of c-Kit as a marker of cardiac stem cells, and role(s) of c-Kit+ cells in myocardial development or in response to pathologic injury remain unresolved due to varied findings among investigators and experimental model systems. Alternative experimental models and approaches are needed to achieve a broader perspective of cardiac c-Kit biology that contextualizes discrepant published observations. Objectives: Tracking c-Kit expression using transgenesis overcomes limitations inherent to knock-in reporter models. Two novel, inducible transgenic c-Kit reporter models are presented in this study to further elaborate on myocardial c-Kit biology. Methods: A previously characterized mouse c-Kit promoter segment was engineered to generate a transgenic mouse in which rtTA transactivator is expressed in c-Kit+ cells (c-KitrtTA). c-KitrtTA crossed to Tet-Responsive-Element(TRE)-Histone2B-EGFP or TRE-Cre lines produces the CKH2B and CKCre double transgenic lines, which express doxycycline-inducible H2BEGFP or Cre proteins in c-Kit+ cells. The CKmTmG triple transgenic mouse, arising from CKCre crossed to the ROSAmTmG reporter line, utilizes doxycycline induced recombination to tag c-Kit+ cells irreversibly with membrane bound EGFP. Endogenous c-Kit and transgenic reporter expression was assessed in adult cardiac myocyte and nonmyocyte cells from these mice under resting and cellular stress conditions using immunohistochemistry and flow cytometry. Results: Coincidence of c-Kit and EGFP is observed in approximately 75% of freshly isolated nonmyocyte cells as detected by flow cytometry. A subpopulation of cardiomyocytes express H2BEGFP or mEGFP in the uninjured, doxycycline treated adult heart. H2BEGFP and c-Kit expression increase in myocytes in response to isoproterenol-induced pathologic stress in vivo and in vitro. Conclusion: These c-Kit transgenic reporter models provide sensitive, specific, inducible and persistent tracking of c-Kit promoter activation. Results presented here reveal an unexpected role for c-Kit expression in adult cardiomyocytes. Future studies will use both models to investigate c-Kit expression in all cell types during cardiac formation and repair.

Download Full-text