Analyzing muscle structure and function throughout the larval instars in live Drosophila

Balakrishnan, Mridula; Sisso, Whitney J.; Baylies, Mary K.

doi:10.1016/j.xpro.2020.100291

Cited by 9 publications

(5 citation statements)

References 18 publications

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“…For larval muscle detachment analysis desired genotypes were crossed into a fluorescent Z-disc marker (α-actinin-GFP). Resulting larvae were then heat-fixed by submerging them into 65°C PBS for 1–2 s and mounted in halo carbon before immediately being imaged [ 76 ].…”

Section: Methodsmentioning

confidence: 99%

The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion

Camp,

Venkatesh,

Solianova

et al. 2024

PLoS Genet

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Cell adhesion requires linkage of transmembrane receptors to the cytoskeleton through intermediary linker proteins. Integrin-based adhesion to the extracellular matrix (ECM) involves large adhesion complexes that contain multiple cytoskeletal adapters that connect to the actin cytoskeleton. Many of these adapters, including the essential cytoskeletal linker Talin, have been shown to contain multiple actin-binding sites (ABSs) within a single protein. To investigate the possible role of having such a variety of ways of linking integrins to the cytoskeleton, we generated mutations in multiple actin binding sites in Drosophila talin. Using this approach, we have been able to show that different actin-binding sites in talin have both unique and complementary roles in integrin-mediated adhesion. Specifically, mutations in either the C-terminal ABS3 or the centrally located ABS2 result in lethality showing that they have unique and non-redundant function in some contexts. On the other hand, flies simultaneously expressing both the ABS2 and ABS3 mutants exhibit a milder phenotype than either mutant by itself, suggesting overlap in function in other contexts. Detailed phenotypic analysis of ABS mutants elucidated the unique roles of the talin ABSs during embryonic development as well as provided support for the hypothesis that talin acts as a dimer in in vivo contexts. Overall, our work highlights how the ability of adhesion complexes to link to the cytoskeleton in multiple ways provides redundancy, and consequently robustness, but also allows a capacity for functional specialization.

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Section: Methodsmentioning

confidence: 99%

The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion

Camp,

Venkatesh,

Solianova

et al. 2024

PLoS Genet

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“…Movies were converted into image sequences (1 image per second) and each larva was tracked using the Manual Tracking plug-in on Fiji. Average velocity was calculated as in (Balakrishnan et al, 2021). For each genotype, we performed three replicates with ~10 larvae each.…”

Section: Larval Trackingmentioning

confidence: 99%

“…Muscle length, sarcomere size and number were quantified as described in step 32 of (Balakrishnan et al, 2021). For accuracy and to assess potential regional differences, sarcomere size was also quantified by an alternative method.…”

Section: Sarcomere and Thin Filament Size/count Analysismentioning

confidence: 99%

Drosophila Tropomodulin is required for multiple actin-dependent processes in myofiber assembly and maintenance

Morales

Carman

Soffar

et al. 2022

Preprint

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Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. While the protein components of myofibrils are generally known, less is known about the mechanisms by which they individually function and together synergize for myofibril assembly and maintenance. For example, it is unclear how the disruption of actin filament (F-actin) regulatory proteins leads to the muscle weakness observed in myopathies. Here, we show that knockdown of Drosophila Tropomodulin (Tmod), a F-actin pointed-end capping protein, results in several myopathy-related phenotypes, including reduction of muscle cell (myofiber) size, increased sarcomere length, disorganization and misorientation of myofibrils, ectopic F-actin accumulation, loss of tension-mediating proteins at the myotendinous junction, and misshaped and internalized nuclei. Our findings support and extend the tension-driven self-organization myofibrillogenesis model. We show that, like its mammalian counterpart, Drosophila Tmod caps filament pointed ends, and this activity is critical for cellular processes in different locations within the myofiber that directly and indirectly contribute to the maintenance of muscle function. Our findings provide significant insights to the role of Tmod in muscle development, maintenance, and disease.

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“…The transcriptional regulators that drive muscle cell differentiation are similar between Drosophila and humans ( Tapscott et al, 1988 ; Maire et al, 2020 ). In both species, differentiated myoblasts fuse to form multinucleated muscle fibers that attach to tendon cells ( Ovalle, 1987 ; Krimm et al, 2002 ; Schulman et al, 2015 ; Lemke & Schnorrer, 2018 ; Richier et al, 2018 ; Balakrishnan et al, 2021 ). Thus, much of muscle development and physiology is shared between the two species.…”

Section: Introductionmentioning

confidence: 99%

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

Shaw

Rios-Monterrosa

Fedorchak

et al. 2022

Front. Cell Dev. Biol.

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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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Analyzing muscle structure and function throughout the larval instars in live Drosophila

Cited by 9 publications

References 18 publications

The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion

The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion

Drosophila Tropomodulin is required for multiple actin-dependent processes in myofiber assembly and maintenance

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

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