Effects of mutant lamins on nucleo-cytoskeletal coupling in Drosophila models of LMNA muscular dystrophy

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.

] Supplemental Figure 4: Specific mutant versions of LamC alter the localization of FG-containing nuclear pore proteins. Body wall muscles from larvae expressing either wild type or mutant LamC were stained with antibodies to LamC (orange) or FG-repeat containing nuclear pore proteins (NUPs) (green), phalloidin (magenta), and DAPI (blue). Staining with antibodies that recognize FG-repeat containing NUPs showed that specific mutant lamins (R237P, R205W, G489V, K521Q, and R564P) caused cytoplasmic aggregations of NUPs (yellow arrows). Other mutant versions of LamC (DK47 and L74R) caused nuclear aggregation of FG-repeat containing NUPs (yellow arrowheads). K521Q also showed perinuclear aggregation of NUPs (plus sign). Scale Bar: 30 μm. Figure 5: Mutant LamC does not alter the localization of alpha-actinin in larval body wall muscles. Larvae expressing either wild-type or mutant LamC were dissected and stained with antibodies to alpha-actinin (green), DAPI (blue) and phalloidin (magenta). In all cases examined, alphaactinin was observed to exhibit the herringbone pattern consistent with proper localization to Z-bands. Figure 6: Specific mutant versions of LamC alter larval motility. The average velocity was calculated and plotted for ten larvae of each genotype studied. A one-way ANOVA was used to determine significance differences among genotypes. *, p ≤ 0.05; **, p ≤ 0.01; ***, ≤ 0.001; ****, p ≤ 0.0001 Supplemental Figure 7: Expression of RNAi against LINC complex components provides effective protein knock-down in muscle. Larval body wall muscles expressing transgenes encoding RNAi against LINC complex components were stained with antibodies to the LINC complex proteins (green) and DAPI (blue). Expression of an RNAi against a non-specific control (Luciferase) did not disrupt localization of Koi, Msp300, and Klar. Expression of an RNAi against Koi caused the anticipated loss of Koi from the nuclear envelope and no change in the localization of Msp300 and Klar. Expression of an RNAi against Msp300 caused the anticipated loss of Msp300 from the nuclear envelope but did not alter Klar localization. Expression of an RNAi against Klar showed the anticipated loss of Klar from the nuclear envelope with no change in the localization of Msp300 and Koi. Scale bar represents 30 mm. Figure 8: Muscle-specific depletion of Msp300 causes reduced larval motility. The average velocity was calculated and plotted for ten larvae of each genotype studied. A one-way ANOVA was used to determine significance differences among genotypes. ***, ≤ 0.001 Supplemental Figure 9. Nuclear envelope localization of koi and Msp300 is maintained upon expression of LamC DK47 and K521Q. Larval body wall muscles expressing either wild-type LamC, DK47 or K521Q were stained with phalloidin (magenta), DAPI (blue) and antibodies to koi or Msp300 (green). The localization of koi and Msp300 was not overtly perturbed in these genetic backgrounds. Scale bar indicates 10 µm.

Supplemental Video 1. Microharpooning of larval body wall muscles expressing wild-type LamC.
The microharpoon (visualized on the right) was inserted into the cytoskeleton ~10 to15 μm from the edge of the nucleus and pulled 30 μm in the direction away from the nucleus at a rate of two μm/s using custom MATLAB software to control the motorized micromanipulator (Eppendorf InjectMan NI2). Pull direction was along the long axis of the myofiber and away from the nucleus. Images were acquired at 32× magnification (20× objective with 1.6× Optivar) every five seconds.
Supplemental Video 2. Microharpooning of larval body wall muscles expressing LamC L74R. The microharpoon was inserted into the cytoskeleton ~10 to15 μm from the edge of the nucleus (visualized on the right) and pulled 30 μm in the direction away from the nucleus at a rate of two μm/s using custom MATLAB software to control the motorized micromanipulator (Eppendorf InjectMan NI2). Pull direction was along the long axis of the myofiber and away from the nucleus. Images were acquired at 32× magnification (20× objective with 1.6× Optivar) every five seconds. Note the increased nuclear deformation relative to the control in video 1.

Supplemental Video 3. Microhaprooning of larval body wall muscles expressing LamC K521Q.
The microharpoon was inserted into the cytoskeleton ~10 to15 μm from the edge of the nucleus (based on crosshairs in the objective) and pulled 30 μm in the direction away from the nucleus at a rate of two μm/s using custom MATLAB software to control the motorized micromanipulator (Eppendorf InjectMan NI2). Pull direction was along the long axis of the myofiber and away from the nucleus. Images were acquired at 32× magnification (20× objective with 1.6× Optivar) every five seconds. Note the lack of centroid displacement compared to the control in video 1.

Supplemental Video 4. Microharpooning of larval body wall muscles expressing LamC R564P.
The microharpoon was inserted into the cytoskeleton ~10 to15 μm from the edge of the nucleus (based on crosshairs in the objective) and pulled 30 μm in the direction away from the nucleus at a rate of two μm/s using custom MATLAB software to control the motorized micromanipulator (Eppendorf InjectMan NI2). Pull direction was along the long axis of the myofiber and away from the nucleus. Images were acquired at 32× magnification (20× objective with 1.6× Optivar) every five seconds. Note the similarities in nuclear deformation and centroid displacement to that of the control in video 1.

Supplemental Video 5. Microharpooning of larval body wall muscles expressing an RNAi against
Luciferase as a control. The microharpoon was inserted into the cytoskeleton ~10 to15 μm from the edge of the nucleus (based on crosshairs in the objective) and pulled 30 μm in the direction away from the nucleus at a rate of two μm/s using custom MATLAB software to control the motorized micromanipulator (Eppendorf InjectMan NI2). Pull direction was along the long axis of the myofiber and away from the nucleus. Images were acquired at 32× magnification (20× objective with 1.6× Optivar) every five seconds. Supplemental Video 6. Microharpooning of larval body wall muscles depleted of Koi. The microharpoon was inserted into the cytoskeleton ~10 to15 μm from the edge of the nucleus (based on crosshairs in the objective) and pulled 30 μm in the direction away from the nucleus at a rate of two μm/s using custom MATLAB software to control the motorized micromanipulator (Eppendorf InjectMan NI2). Pull direction was along the long axis of the myofiber and away from the nucleus. Images were acquired at 32× magnification (20× objective with 1.6× Optivar) every five seconds. Note that the nuclear deformation and centroid displacement is similar to that of the control in video 5. Supplemental Video 7. Microharpooning of larval body wall muscles depleted of Msp300. The microharpoon was inserted into the cytoskeleton ~10 to15 μm from the edge of the nucleus (based on crosshairs in the objective) and pulled 30 μm in the direction away from the nucleus at a rate of two μm/s using custom MATLAB software to control the motorized micromanipulator (Eppendorf InjectMan NI2). Pull direction was along the long axis of the myofiber and away from the nucleus. Images were acquired at 32× magnification (20× objective with 1.6× Optivar) every five seconds. Note the lack of centroid displacement compared to that of the control in video 5.