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Primary Human MSCs subjected to 1 hr intense cyclic tensile strain with/without 24 hr recovery

ABSTRACT: Cells resident in tissues must be resilient to the physical demands of their surroundings. Our current understanding of cellular mechano-signalling is largely based on static systems, but these models do not reproduce the dynamic nature of living tissue. Here, we examined the time-resolved response of primary human mesenchymal stem cells (hMSCs) to periods of cyclic tensile strain (CTS). We observed parallels between morphological changes following low-intensity strain (1 hour, 4% CTS at 1 Hz) and responses to increased substrate stiffness. However, as the strain regime was intensified (CTS at ≥ 2 Hz), we characterised a broad, structured and reversible protein-level response, even as transcription was apparently shut down. Regulation of the linker of nucleo- and cytoskeleton (LINC) complex proteins, and specifically of SUN domain-containing protein 2 (SUN2), was found to decouple mechano-transmission within the cell and hence isolate the nucleus from cellular deformation.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Multipotent Stem Cell, Bone Marrow

SUBMITTER: Venkatesh Mallikarjun

LAB HEAD: Joe Swift

PROVIDER: PXD012949 | Pride | 2019-04-24

REPOSITORIES: Pride

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Nuclear decoupling is part of a rapid protein-level cellular response to high-intensity mechanical loading.

Gilbert Hamish T J HTJ Mallikarjun Venkatesh V Dobre Oana O Jackson Mark R MR Pedley Robert R Gilmore Andrew P AP Richardson Stephen M SM Swift Joe J

Nature communications 20190912 1

Studies of cellular mechano-signaling have often utilized static models that do not fully replicate the dynamics of living tissues. Here, we examine the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 h, 4% CTS at 1 Hz), cell characteristics mimic responses to increased substrate stiffness. As the strain regime is intensified (frequency increased to 5 Hz), we characterize rapid establishment of a broad, structured ...[more]

PMID: 31515493

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Project description:The chromatin of individual chromosomes is organized into chromosome territories (CTs) in the interphase nucleus. The spatial arrangement of CTs is non-random and evolutionarily conserved. The gene-dense and gene-poor CTs are positioned in the nuclear center and periphery, respectively. As candidates for key molecules involved in nuclear organization, we have investigated the nuclear actin-related proteins (Arps), which include the evolutionarily conserved actin-family together with conventional actin. We used a conditional knockout system with chicken DT40 cells to analyze the functions of the actin-related protein Arp6. Consistent with a previous identification of Arp6 in the SRCAP chromatin remodeling complex, the histone variant H2AZ was significantly decreased in the chromatin of Arp6-deficient cells. Most importantly, Arp6-deficient cells had impaired radial positioning of both gene-poor macrochromosome and gene-rich microchromosome CTs. A transcription microarray analysis of the cells suggests that the radial positioning of CTs impacts only a limited number of genes and plays an active role in repression, rather than in induction. As far as we know, this report is the first observation that an inner nuclear protein is required for the radial distribution of CTs, and will provide new insight into the mechanisms and physical significance of the positioning of CTs in the nucleus. The total RNA was extracted with RNeasy Maxi Kit (QIAGEN) according to the manufacturer protocol. The poly-A mRNA was isolated from total RNA using Oligotex-dT30 Super (JSR corporation) and biotinylated cRNA was synthesized according to Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix). Shortly, 2 ug of poly-A mRNA was reverse transcribed to cDNA using 100 pmol of a T7-Oligo (dT) Primer (Invitrogen), and biotinylated cRNA was prepared by in vitro transcription amplification. For hybridization, 15 ug of biotinylated cRNA was fragmented and added into a hybridization cocktail containing four biotinylated hybridization controls (bioB, bioC, bioD, and cre) as recommended by the manufacturer. GeneChip Chicken genome Arrays (Affymetrix) were hybridized with the cocktail at 45ËC for 16 hours. After washing with Non-Stringent wash buffer (6xSSPE, 0.01% Tween20) and staining with SAPE in the Affymetrix GeneChip Fluidics Station 400, the GeneChips were read using the Affymetrix GeneChip scanner 3000 7G. These systems and data analyses were operated with the GeneChip Operating Software 1.3.

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