The nuclei of multinucleated skeletal muscles experience substantial external force during development and muscle contraction. Protection from such forces is partly provided by lamins, intermediate filaments that form a scaffold lining the inner nuclear membrane. Lamins play a myriad of roles, including maintenance of nuclear shape and stability, mediation of nuclear mechanoresponses, and nucleo-cytoskeletal coupling. Herein, we investigate how disease-causing mutant lamins alter myonuclear properties in response to mechanical force. This was accomplished via a novel application of a micropipette harpooning assay applied to larval body wall muscles of Drosophila models of lamin-associated muscular dystrophy. The assay enables the measurement of both nuclear deformability and intracellular force transmission between the cytoskeleton and nuclear interior in intact muscle fibers. Our studies revealed that specific mutant lamins increase nuclear deformability while other mutant lamins cause nucleo-cytoskeletal coupling defects, which were associated with loss of microtubular nuclear caging. We found that microtubule caging of the nucleus depended on Msp300, a KASH domain protein that is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Taken together, these findings identified residues in lamins required for connecting the nucleus to the cytoskeleton and suggest that not all muscle disease-causing mutant lamins produce similar defects in subcellular mechanics.
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Data‐Driven and Cell‐Specific Determination of Nuclei‐Associated Actin Structure
Quantitative volumetric assessment of filamentous actin (F‐actin) fibers remains challenging due to their interconnected nature, leading researchers to utilize threshold‐based or qualitative measurement methods with poor reproducibility. Herein, a novel machine learning‐based methodology is introduced for accurate quantification and reconstruction of nuclei‐associated F‐actin. Utilizing a convolutional neural network (CNN), actin filaments and nuclei from 3D confocal microscopy images are segmented and then each fiber is reconstructed by connecting intersecting contours on cross‐sectional slices. This allows measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F‐actin in supporting nucleocytoskeletal connectivity, apical F‐actin, basal F‐actin, and nuclear architecture in mesenchymal stem cells (MSCs) are quantified following the disruption of the linker of nucleoskeleton and cytoskeleton (LINC) complexes. Disabling LINC in MSCs generates F‐actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. The findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F‐actin.
more » « less- NSF-PAR ID:
- 10491237
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Small Structures
- ISSN:
- 2688-4062
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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