Septin 7 Interacts With Numb To Preserve Sarcomere Structural Organization And Muscle Contractile Function

Reviewer #1 (Public Review):

This work investigates the function of the PTB domain containing adaptor protein Numb in skeletal muscle structure and function. In particular, the effects of reduced Numb expression in aging muscle is proposed as a mechanism for reduced contractile function associated with sarcopenia. Using ex-vivo analysis of conditional Numb and Numblike knockout muscle the authors demonstrate that loss of Numb but not the related Numblike expression perturbs muscle muscle force generation. In order to explore the molecular mechanisms involved, Numb interacting proteins were identified in C2C12 cell cultured myotubes by immunoprecipitation and LC-MS/MS. The authors identify Septin 7 as a Numb binding protein and demonstrate that loss of Numb/Numblike in myofibers causes changes in Septin 7 subcellular localization. Several questions remain. The authors could provide further clarity on the expression of Numb and Numb-like proteins and the specificity of antibodies used in this study since some Numb antibodies recognize both Numb and Numblike. The authors focus on septin 7 amongst the list of potential Numb interactions identified by AP-MS. Of note, septin 2, 9 and 10 were also identified in the AP-MS experiment. Whether these septins form a complex or are also disrupted by Numb/Numblike loss remains an interesting area for further investigation. Additional investigation of the specificity and mapping of the Numb-Septin 7 (or another Septin) interaction would be of interest and provide an approach for future studies to demonstrate the biological relevance and specificity of the Numb-Septin 7 interaction in skeletal muscle.

Reviewer #2 (Public Review):

Summary:
This main purpose of this investigation was to 1) compare the effects of a single knockout (sKO) of Numb or a double knockout (dKO) of Numb and NumbL on ex-vivo physiological properties of the extensor digitorium longus (EDL) muscle in C57BL/6NCrl mice; and 2) analyze protein complexes isolated from C2C12 myotubes via immunoprecipitation and LC/MS/MS for potential Numb binding partners. The main findings are 1) the muscles from sKO and dKO were significantly weaker with little difference between the sKO and dKO lines, indicating the reduced force is mainly due to the inactivation of the Numb gene; and 2) there were 11 potential Numb binding proteins that were identified and cytoskeletal specific proteins including Septin 7.

Strengths:
Strait-forward yet elegant design to help determine the important role the Numb has in skeletal muscle.

Weaknesses: There were a limited number of samples (3-6) that were used for the physiological experiments; however, there was a very large effect size in terms of differences in muscle tension development between the induced KO models and the controls.