Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

AbstractCentriolar satellites are dynamic, membrane-less granules composed of over 200 proteins. They store, modify, and traffic centrosome and primary cilium proteins, and help to regulate both the biogenesis and some functions of centrosomes and cilium. In most cell types, satellites cluster around the perinuclear centrosome, but their integrity and cellular distribution are dynamically remodeled in response to different stimuli, such as cell cycle cues. Dissecting the specific and temporal functions and mechanisms of satellites and how these are influenced by their cellular positioning and dynamics has been challenging using genetic approaches, particularly in ciliated and proliferating cells. To address this, we developed a chemical-based trafficking assay to rapidly and efficiently redistribute satellites to either the cell periphery or center, and fuse them into stable clusters in a temporally-controlled way. Induced satellite clustering at either the periphery or center resulted in antagonistic changes in the pericentrosomal levels of a subset of proteins, revealing a direct role for their positioning in protein targeting and sequestration. Systematic analysis of the interactome of peripheral satellite clusters revealed enrichment of proteins implicated in cilium biogenesis and mitosis. Importantly, inducible peripheral satellite targeting in ciliated cells revealed a function for satellites not just for efficient cilium assembly, but also in the maintenance of steady-state cilia and in cilia disassembly by regulating the structural integrity of the ciliary axoneme. Finally, although perturbing satellite distribution and dynamics inhibited their mitotic dissolution, it did not cause mitotic defects. Collectively, our results for the first time showed a direct link between satellite functions and their pericentrosomal clustering, and provided a new tool for probing temporal satellite functions in different contexts.

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Physical modelling of failure in composites

Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rsta.2015.0280 ◽

2016 ◽

Vol 374 (2071) ◽

pp. 20150280 ◽

Cited By ~ 17

Author(s):

Ramesh Talreja

Keyword(s):

Composite Materials ◽

Failure Modes ◽

Structural Integrity ◽

Failure Mechanisms ◽

Local Stress ◽

Physical Modelling ◽

Stress Fields ◽

Systematic Analysis ◽

Composite Failure ◽

Failure Theories

Structural integrity of composite materials is governed by failure mechanisms that initiate at the scale of the microstructure. The local stress fields evolve with the progression of the failure mechanisms. Within the full span from initiation to criticality of the failure mechanisms, the governing length scales in a fibre-reinforced composite change from the fibre size to the characteristic fibre-architecture sizes, and eventually to a structural size, depending on the composite configuration and structural geometry as well as the imposed loading environment. Thus, a physical modelling of failure in composites must necessarily be of multi-scale nature, although not always with the same hierarchy for each failure mode. With this background, the paper examines the currently available main composite failure theories to assess their ability to capture the essential features of failure. A case is made for an alternative in the form of physical modelling and its skeleton is constructed based on physical observations and systematic analysis of the basic failure modes and associated stress fields and energy balances. This article is part of the themed issue ‘Multiscale modelling of the structural integrity of composite materials’.

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Cellular Functions of OCT-3/4 Regulated by Ubiquitination in Proliferating Cells

Cancers ◽

10.3390/cancers12030663 ◽

2020 ◽

Vol 12 (3) ◽

pp. 663

Author(s):

Kwang-Hyun Baek ◽

Jihye Choi ◽

Chang-Zhu Pei

Keyword(s):

Stem Cells ◽

Cellular Proliferation ◽

Critical Role ◽

Embryonic Stem ◽

Cell Types ◽

Ubiquitin Proteasome System ◽

Specific Cell ◽

Cellular Mechanisms ◽

Proliferating Cells ◽

Proliferation And Differentiation

Octamer-binding transcription factor 3/4 (OCT-3/4), which is involved in the tumorigenesis of somatic cancers, has diverse functions during cancer development. Overexpression of OCT-3/4 has been detected in various human somatic tumors, indicating that OCT-3/4 activation may contribute to the development and progression of cancers. Stem cells can undergo self-renewal, pluripotency, and reprogramming with the help of at least four transcription factors, OCT-3/4, SRY box-containing gene 2 (SOX2), Krüppel-like factor 4 (KLF4), and c-MYC. Of these, OCT-3/4 plays a critical role in maintenance of undifferentiated state of embryonic stem cells (ESCs) and in production of induced pluripotent stem cells (iPSCs). Stem cells can undergo partitioning through mitosis and separate into specific cell types, three embryonic germ layers: the endoderm, the mesoderm, and the trophectoderm. It has been demonstrated that the stability of OCT-3/4 is mediated by the ubiquitin-proteasome system (UPS), which is one of the key cellular mechanisms for cellular homeostasis. The framework of the mechanism is simple, but the proteolytic machinery is complicated. Ubiquitination promotes protein degradation, and ubiquitination of OCT-3/4 leads to regulation of cellular proliferation and differentiation. Therefore, it is expected that OCT-3/4 may play a key role in proliferation and differentiation of proliferating cells.

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Single-cell transcriptomics reveals immune response of intestinal cell types to viral infection

10.1101/2020.08.19.255893 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sergio Triana ◽

Megan L. Stanifer ◽

Mohammed Shahraz ◽

Markus Mukenhirn ◽

Carmon Kee ◽

...

Keyword(s):

Immune Response ◽

Single Cell ◽

Cell Lineage ◽

Enteric Virus ◽

Cell Types ◽

Intestinal Cell ◽

Intestinal Epithelial ◽

Proliferating Cells ◽

Interferon Stimulated Genes ◽

Human Intestinal Epithelium

AbstractHuman intestinal epithelial cells form a primary barrier protecting us from pathogens, yet only limited knowledge is available about individual contribution of each cell type to mounting an immune response against infection. Here, we developed a pipeline combining single-cell RNA-Seq and highly-multiplex RNA imaging and applied it to human intestinal organoids infected with human astrovirus, a model human enteric virus. We found that interferon controls the infection and that astrovirus infects all major cell types and lineages with a preferential infection of proliferating cells. Intriguingly, each intestinal epithelial cell lineage has a unique basal expression of interferon-stimulated genes and, upon astrovirus infection, undergoes an antiviral transcriptional reprogramming by upregulating distinct sets of interferon-stimulated genes. These findings suggest that in the human intestinal epithelium, each cell lineage plays a unique role in resolving virus infection. Our pipeline can be applicable to other organoids and viruses, opening new avenues to unravel roles of individual cell types in viral pathogenesis.

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AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A

10.1101/235226 ◽

2017 ◽

Cited By ~ 1

Author(s):

Alexandra K. Davies ◽

Daniel N. Itzhak ◽

James R. Edgar ◽

Tara L. Archuleta ◽

Jennifer Hirst ◽

...

Keyword(s):

Membrane Trafficking ◽

Close Association ◽

Adaptor Protein ◽

Cell Types ◽

Subcellular Localisation ◽

Cell Periphery ◽

Accessory Proteins ◽

Unknown Aetiology ◽

Spatial Control ◽

Cargo Proteins

AbstractAdaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including ‘Dynamic Organellar Maps’, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the “ATG9A reservoir” required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

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STUDIES ON CELL DEFORMABILITY

The Journal of Cell Biology ◽

10.1083/jcb.33.2.341 ◽

1967 ◽

Vol 33 (2) ◽

pp. 341-347 ◽

Cited By ~ 20

Author(s):

Leonard Weiss

Keyword(s):

Cell Types ◽

Mechanical Effect ◽

Cell Periphery ◽

Detectable Change ◽

Anionic Sites ◽

Cell Deformability ◽

Edta Treatment ◽

Edta Solution ◽

Hydrodynamic Slip ◽

Sarcoma 37

The nonlethal procedure of incubation in EDTA solution makes the peripheral regions of ascites sarcoma 37 cells more easily deformable, as reflected in measurements of the decreased amount of negative pressure required to suck out standard hemispherical bulges from the cells into micropipettes. The facilitation of deformability was abolished after reincubation of cells in calcium-containing saline, and this mechanical parameter was partially restored to normal after reincubation in magnesium-containing saline; the mechanical effect of EDTA treatment is, therefore, thought to be due mainly to the removal of calcium from the cell periphery. As EDTA treatment produces no detectable change in cellular electrophoretic mobility, it is concluded that peripheral calcium must be bound to anionic sites deeper than about 10 A from the cellular hydrodynamic slip plane. The data are discussed with emphasis on the view that they should not be extrapolated freely to other cell types.

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Profiling proliferative cells and their progeny in damaged murine hearts

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805829115 ◽

2018 ◽

Vol 115 (52) ◽

pp. E12245-E12254 ◽

Cited By ~ 48

Author(s):

Kai Kretzschmar ◽

Yorick Post ◽

Marie Bannier-Hélaouët ◽

Andrea Mattiotti ◽

Jarno Drost ◽

...

Keyword(s):

Stem Cell ◽

Cell Function ◽

Cardiac Fibroblasts ◽

Cardiac Injury ◽

Cardiac Regeneration ◽

Cell Types ◽

Postnatal Growth ◽

Lineage Tracing ◽

Genetic Lineage ◽

Proliferating Cells

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.

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A Fresh Look at the Structure, Regulation, and Functions of Fodrin

Molecular and Cellular Biology ◽

10.1128/mcb.00133-20 ◽

2020 ◽

Vol 40 (17) ◽

Cited By ~ 1

Author(s):

Jamuna S. Sreeja ◽

Rince John ◽

Dhrishya Dharmapal ◽

Rohith Kumar Nellikka ◽

Suparna Sengupta

Keyword(s):

Posttranslational Modifications ◽

Mammalian Cells ◽

Structural Integrity ◽

Cell Function ◽

Cell Types ◽

Erythroid Cell ◽

Structural Variations ◽

Neuronal Tissues ◽

Different Cell Types ◽

Neuronal Disorders

ABSTRACT Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αβ) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.

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The LIM Domain Protein Lmo4 Is Highly Expressed in Proliferating Mouse Epithelial Tissues

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4a6553.2005 ◽

2005 ◽

Vol 53 (4) ◽

pp. 475-486 ◽

Cited By ~ 31

Author(s):

Eleanor Y.M. Sum ◽

Lorraine A. O'Reilly ◽

Nadeen Jonas ◽

Geoffrey J. Lindeman ◽

Jane E. Visvader

Keyword(s):

Small Intestine ◽

Mammary Gland ◽

Cell Types ◽

Mammary Epithelial ◽

Specific Cell ◽

Basal Cells ◽

Proliferating Cells ◽

Terminal End Buds ◽

Mouse Tissues ◽

Important Regulator

LMO4 belongs to the LIM-only family of zinc finger proteins that have been implicated in oncogenesis. The LMO4 gene is overexpressed in breast cancer and oral cavity carcinomas, and high levels of this protein inhibit mammary epithelial differentiation. Targeted deletion of Lmo4 in mice leads to complex phenotypic abnormalities and perinatal lethality. To further understand the role of LMO4, we have characterized Lmo4 expression in adult mouse tissues by immunohistochemical staining using monoclonal anti-Lmo4 antibodies. Lmo4 was highly expressed within specific cell types in diverse tissues. Expression was prevalent in epithelial-derived tissues, including the mammary gland, tongue, skin, small intestine, lung, and brain. High levels of Lmo4 were frequently observed in proliferating cells, such as the crypt cells of the small intestine and the basal cells of the skin and tongue. Lmo4 was highly expressed in the proliferative cap cell layer of the terminal end buds in the peripubertal mammary gland and in the lobuloalveolar units during pregnancy. The expression profile of Lmo4 suggests that this cofactor is an important regulator of epithelial proliferation and has implications for its role in the pathogenicity of cancer.

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Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces

International Journal of Molecular Sciences ◽

10.3390/ijms20246221 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6221 ◽

Cited By ~ 4

Author(s):

Jutamas Uttagomol ◽

Usama Sharif Ahmad ◽

Ambreen Rehman ◽

Yunying Huang ◽

Ana C. Laly ◽

...

Keyword(s):

Target Genes ◽

Hippo Pathway ◽

Cyclic Strain ◽

Cell Periphery ◽

Mechanical Forces ◽

Force Transmission ◽

Direct Role ◽

The Impact ◽

Desmoglein 3 ◽

E Cadherin

Desmoglein 3 (Dsg3) plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction in keratinocytes. A direct comparison was made with E-cadherin, a well-characterized mechanosensor. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in the expression and localization of junction assembly proteins. The knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of yes-associated protein (YAP), a mechanosensory, and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 formed a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to the strain or substrate rigidity-induced nuclear relocation of YAP and phospho-YAP. Plakophilin 1 (PKP1) seemed to be crucial in recruiting the complex containing Dsg3/phospho-YAP to the cell surface since its silencing affected Dsg3 junctional assembly with concomitant loss of phospho-YAP at the cell periphery. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes and cell proliferation. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

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Constitutive Genetic Deletion of Hcn1 Increases Alcohol Preference during Adolescence

Brain Sciences ◽

10.3390/brainsci10110763 ◽

2020 ◽

Vol 10 (11) ◽

pp. 763

Author(s):

Michael C. Salling ◽

Neil L. Harrison

Keyword(s):

Prefrontal Cortex ◽

Alcohol Consumption ◽

Medial Prefrontal Cortex ◽

Neuronal Excitability ◽

Cell Types ◽

Brain Regions ◽

Alcohol Preference ◽

Hcn Channels ◽

Direct Role ◽

Genetic Deletion

The hyperpolarization-activated cyclic nucleotide-gated channel (HCN), which underlies the hyperpolarization-activated cation current (Ih), has diverse roles in regulating neuronal excitability across cell types and brain regions. Recently, HCN channels have been implicated in preclinical models of substance abuse including alcohol. In the prefrontal cortex of rodents, HCN expression and Ih magnitude are developmentally regulated during adolescence and may be vulnerable to alcohol’s effects. In mice, binge alcohol consumption during the adolescent period results in a sustained reduction in Ih that coincides with increased alcohol consumption in adulthood, yet the direct role HCN channels have on alcohol consumption are unknown. Here, we show that the genetic deletion of Hcn1 causes an increase in alcohol preference on intermittent 2-bottle choice task in homozygous null (HCN1−/−) male mice compared to wild-type littermates without affecting saccharine or quinine preference. The targeted viral deletion of HCN1 in pyramidal neurons of the medial prefrontal cortex resulted in a gradual loss of Hcn1 expression and a reduction in Ih magnitude during adolescence, however, this did not significantly affect alcohol consumption or preference. We conclude that while HCN1 regulates alcohol preference, the genetic deletion of Hcn1 in the medial prefrontal cortex does not appear to be the locus for this effect.

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