Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein

Abstract

Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active, microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use, a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.

Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation

“…Since Mbands do not form in the homozygous targeted cells, it is unlikely that myosin will become organised into muscle sarcomeres, even though the A-band region of titin -which contains over 170 domains that can bind to myosin (Labeit et al, 1992) -remains intact. In addition, the incorporation of myosin into nascent sarcomeres might require the microtubule network (Pizon et al, 2002 and references therein), with which MURF2 and MURF3 also interact. Myofibrils in neonatal rat cardiomyocytes are relatively stable when treated with nocodazole, which depolymerises microtubules (RothenRutishauer et al, 1998).…”

“…Myofibrils in neonatal rat cardiomyocytes are relatively stable when treated with nocodazole, which depolymerises microtubules (RothenRutishauer et al, 1998). However, MURF2 preferentially interacts with modified (detyrosinated) microtubules (Pizon et al, 2002), which are relatively resistant to depolymerisation by nocodazole (Gundersen et al, 1994). The deletion of the MURF2-binding site on titin in the targeted cells might contribute to the disrupted assembly of sarcomeric myosin if this interaction is required to regulate microtubule-directed myosin integration with the titin filament.…”

The M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere

“…A pivotal role in the stress sensing might be assumed by the Ser/Thr-kinase domain in the titin M-band portion, which is activated by stretching [55] and provides a binding platform for several proteins involved in signal transduction between sarcomere and nucleus [56]. Other titin-binding proteins in the M-band are DRAL/FHL-2, a member of the LIM-domain family suggested to function as an adaptor for metabolic enzymes [57], and two muscle-specific RING-finger proteins MURF-1 and MURF-2 [58,59], involved in multiple protein interactions. Another protein potentially implicated in stress sensing is Smpx/Csl, which was originally identified as a gene upregulated in passively stretched skeletal muscle fibers [60].…”

Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I

“…MuRF1 interacts with the myofibrillar giant spring protein titin at the M line (13,14), resulting in disruption of the subdomain that binds MuRF1. This interaction suggests that MuRF1 regulates the stability of this large structural protein, although the functional consequences of this interaction remain to be explored, and titin does not appear to be a substrate for the ubiquitin ligase activity of MuRF1.…”