We have identified MTM1 as an IF-binding protein and present evidence herein for MTM1-based regulation of desmin IF and mitochondrial dynamics in muscle cells. MTM1 bound directly to desmin and affected filament assembly in vitro and architecture in vivo. Molecular dissection of the interaction defined the Rac1-induced recruitment domain of MTM1 (22
) and the 2B rod domain of desmin as the molecular interface. Several mutations leading to XLCNM and DRM, respectively, are located to these domains (23
), highlighting the importance of this complex in skeletal muscle. Analysis of the 2B rod domain of desmin has attracted much attention, since a number of desmin mutations occur in this region, and it has previously been implicated in filament assembly (26
). As excess MTM1 led to abnormal shape and branching of desmin filaments in vitro, it is possible that MTM1 binding to desmin affects the structure of newly formed filaments. These abnormal filaments are similar to those formed in the presence of a mutated (R120G) αB-crystallin (27
). This gain-of-function R120G mutation in αB-crystallin also leads to desmin aggregation and to a DRCM (28
). Previous studies indicate that the YRKLLEGEE motif of desmin (DES-403-410) is crucial for the formation of authentic tetrameric complexes and for the control of filament width, rather than elongation, during assembly (29
). MTM1 binding to the DES-352-377 region (adjacent to the YRKLLEGEE motif) may regulate the formation and/or turnover of desmin filaments in muscle cells. Moreover, Colakoglu and Brown proposed a mechanism, termed intercalary subunit exchange
, for vimentin and neurofilaments in cells (30
). They showed that IF subunits incorporate along the length of filaments without compromising interaction among other subunits, leading to a unique turnover and alteration of IF polymer composition. The implication of IF-binding proteins, such as MTM1 or αB-crystallin, in this process remains to be evaluated.
IFs have a well-established role as major mechanical integrators of cells and tissues. This function is mainly attributed to their unique viscoelastic properties that render them more resistant to deformation under mechanical stress (31
). In addition to their unusual viscoelasticity, IFs also exhibit highly dynamic properties in vivo. The concept that the binding of proteins influences MF and/or MT dynamics by exploiting the intrinsic structural plasticity of the filaments currently extends to IFs, as more IF-binding proteins are being discovered. IFs also appear to be involved in the distribution of membrane-bound organelles (32
). Although the transport of membranous organelles is mainly mediated by molecular motors and their respective cytoskeletal tracks, their proper positioning in the cytoplasm frequently appears to involve interactions with IFs. IFs have been recently implicated in organelle transport and particularly in mitochondrial trafficking (33
). They are believed to be involved in anchoring mitochondria at sites of high energy demand or in the vicinity of other organelles to allow for effective signaling. Indeed, loss of desmin (8
) or MTM1 in muscle cells (present study) has been shown to lead to mitochondrial positioning and dynamics defects. IFs may also directly influence mitochondrial membrane behavior by supporting proper protein and lipid targeting. Loss of accurate mitochondrial positioning in desmin-null cardiomyocytes leads to loss of proximity to other organelles, including the myofibrillar contractile apparatus and the endoplasmic/sarcoplasmic reticulum (8
). In Mtm1
-KD and -KO cells, and in cells in which MTM1-desmin interaction is disrupted (overexpression of MTM1 mutants with a dominant-negative effect), both mitochondria and IFs accumulated at the perinuclear region. Mitochondrial collapse was observed even in the case of XLCNM mutations that have maintained their ability to bind desmin, which suggests that MTM1 has a direct, non–desmin-dependent role in mitochondrial dynamics/homeostasis in muscle. In Mtm1
-KD cells, the accumulated mitochondria in the perinuclear compartment were less dynamic compared with mitochondria at the periphery of the cells or in the perinuclear regions of control cells. IF collapse or/and excess of PIs (e.g., PtdIns[3,5]P2
) in the absence of MTM1 may render mitochondria less motile and compromised. In addition, we observed an increased association of desmin to mitochondria in Mtm1
-KO compared with WT muscle. To describe the involvement of MTM1 and desmin in mitochondrial dynamics, 2 distinct — although complementary — mechanisms could be proposed. The first implicates IF-binding proteins, which are able to affect IF dynamics at the structural level. The second implicates plectin as a cytolinker between IFs and mitochondria. Indeed, plectin deficiency causes detachment of desmin IFs from the z-disc, costameres, mitochondria, and nuclei, promoting the formation of desmin aggregates. The potential interconnections between these 2 pathways should be investigated, in particular the functional link among plectins, myotubularin, and αB-crystallin and how they influence desmin filaments.
Several findings support a role for myotubularin in membrane trafficking through its role in PI metabolism (34
). PtdIns3P and PtdIns(3,5)P2
, the substrates of myotubularin, are primarily localized to the membrane of early and late endosomes, respectively, and studies have implicated myotubularin family members in endosomal trafficking events and membrane homeostasis (36
). However, desmin did not affect in vitro or ex vivo MTM1 phosphatase activity, and MTM1 mutants that cannot bind desmin were as enzymatically active as the WT protein (Supplemental Figure 9), which suggests that IFs do not interfere with or modulate MTM1 activity. However, it is possible that desmin plays a role in stabilizing MTM1 association to organelles. MTM1 might have 2 distinct roles. One is to regulate vesicle trafficking and maturation, but also positioning of organelles, such as mitochondria, through the tight regulation of the PI pool in the cell. A second role for MTM1 — likely PI independent — is the regulation of IFs (Figure F).
In normal skeletal muscle, MTM1 colocalized with desmin at the sarcolemma and also at the z-disc. However, in cardiac muscles, no interaction was evident by biochemical approaches, and weaker colocalization was observed for MTM1 and desmin. While desmin mutations have been associated with both cardiomyopathy and myopathy, cardiac involvement is not a common sign of adult XLCNM patients (38
). We also found no alteration in the histology of cardiac muscle in the Mtm1
-KO mice. Together, these observations point to a specific role for the MTM1-desmin complex in skeletal muscle.
Mitochondrial aggregates have been observed both in Des
-KO mice and in DRM biopsies (8
). Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. The observed alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. In addition, transgenic mice carrying the Des
L345P missense mutation are characterized by striking abnormalities in mitochondrial morphology and Ca2+
). Recent findings also demonstrate substantial deregulation of Ca2+
handling and homeostasis in Mtm1
-KO mouse and mtm1
-KD zebrafish muscle, paralleled by the disruption of excitation-contraction coupling linked to T-tubule abnormalities (18
). Combined with our findings of abnormal mitochondrial morphology and positioning in cells expressing MTM1 mutants and in Mtm1
-KD cells, these observations lead us to postulate that deregulation of Ca2+
handling in MTM1 deficiency is caused both by triad disorganization and by a mitochondrial homeostasis defect. As centralization of nuclei is a hallmark of centronuclear myopathy, and as an increased number of internal nuclei is also observed in DRM and Des
-KO mice (24
), we envisage a more general implication of the MTM1-IF complex in organelle positioning.
In summary, we present evidence that MTM1 regulates desmin IF assembly and mitochondrial positioning and dynamics in skeletal muscle cells. We propose that defective mitochondrial homeostasis is a common physiopathological feature of centronuclear myopathies and desmin-related myopathies.