In this paper, we show for the first time cooperative, as well as unique, activities of the two Drosophila KASH proteins MSP-300 and Klar in promoting even spacing and anchoring of myonuclei in striated muscle fibers. A novel MSP-300 nuclear ring assembles and anchors the MTs to the nuclear envelope in a Klar- and MSP-300 KASH–dependent manner, mediating MT astral organization around each nucleus. We suggest that the astral MT associated with each myonucleus, forming a basic unit, which in the steady-state holds each nucleus in place. However, during muscle fiber growth, each unit might change its relative position as a result of MT growth so that the distance between the myonuclei is maintained. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment is mediated exclusively by MSP-300, which maintains physical continuity between the nuclear ring and the Z-discs, presumably through dimerization of the spectrin repeats capable of forming filaments.
Recent results (Metzger et al., 2012
) suggested that reducing KASH proteins from the nuclear membrane (by overexpressing the KASH domain) did not affect nuclear positioning in C2C12 cells. It is possible that residual KASH-dependent activity was present in these cells, capable of rescuing nuclei position.
Our physiological measurements demonstrate the critical contribution of nuclear spacing to larval locomotion and muscle activity, which is consistent with the results of Metzger et al. (2012)
. Significantly, our studies further demonstrate that the contribution of MSP-300 to muscle function is more critical relative to that of Klar (as msp
mutant larvae were significantly slower than klarΔ1–18
mutant larvae), presumably because MSP-300 affects the positioning of the myonuclei as well as that of the mitochondria and ER.
The MT network appears to possess the dynamic properties required for myonuclear organization. Consistently, recent data show the active involvement of opposing MT motors in driving robust nuclear dynamics in differentiating CTC12 cells (Wilson and Holzbaur, 2012
). During embryogenesis, MT arrangement in myotubes is polarized with their plus ends close to the MTJ (Clark et al., 1997
). However, after muscle sarcomerization, the MTs undergo a significant rearrangement into astral organization with their plus ends facing the nuclei (shown by staining with EB1; ). This suggests that nuclear positioning and spacing differs mechanistically between early embryonic and larval stages, i.e., before and after the establishment of sarcomeric architecture. In support of this model, we show that: (a) Klar is the only KASH protein required for nuclear positioning in embryonic myotubes, whereas MSP-300 is dispensable (). (b) The polarity and distribution of the MT network changes during development: in embryos (stage 16), the plus ends are close to the MTJ (Clark et al., 1997
), whereas in striated muscle fibers, the MT forms astral structures surrounding each nucleus with their plus ends facing the nuclear envelope (). (c) The nuclei are arranged in distinct patterns at each stage; e.g., in the embryonic dorsal acute and oblique muscles, the nuclei are positioned in a typical half circle close to the MTJ, whereas in third instar larvae, the nuclei of the same muscles rearrange and are distributed evenly along the entire muscle fiber. Thus, we suggest that sarcomerization involves a significant rearrangement of the MT network and the nuclei, at the end of which the nuclei are spaced evenly along the muscle fiber and attached to the Z-discs.
The mechanism regulating this rearrangement has not been elucidated. We suggest that as part of this rearrangement, MSP-300 is translocated from the MTJ, where it accumulates during embryonic stages (Volk 1992
and unpublished data) into the Z-discs at the end of embryogenesis. Presumably, during this latter stage MSP-300 isoforms containing KASH are produced, and collaborate with Klar to promote the attachment of the astral MT network to the nuclear envelope. Klar function has been previously linked to MTs as a regulator of the MT motors dynein (associated with MT minus ends) and/or kinesin (associated with MT plus ends; Welte et al., 1998
; Mosley-Bishop et al., 1999
; Fischer et al., 2004
; Patterson et al., 2004
; Welte, 2004
). Therefore, Klar function in promoting the association of the MSP-300 nuclear ring and MT plus ends with the nuclear envelope might be mediated through its ability to recruit MT plus-end motors. Nesprin 1 and 2 were also shown to interact with dynein/dynactin and kinesin-1 in developing mouse neurons (Zhang et al., 2009
), and the spectrin repeats region of Nesprin 1 was shown to bind kinesin-2 (Fan and Beck, 2004
). Therefore, Klar can potentially mediate an interaction with MT, which is also consistent with the demonstration that kinesin heavy chain is essential for myonuclei positioning in Drosophila
larval muscles (Metzger et al., 2012
). MSP-300 might be indirectly linked to this activity through its association with Klar.
Because muscle size increases dramatically during larval growth, and the number of nuclei remains constant (Beckett and Baylies, 2006
), it is not clear how nuclear spacing is maintained during this growth process. We suggest that similarly to nuclear spacing in preblastoderm embryos (Baker et al., 1993
), the minus ends of the MT associate with each other, whereas the plus ends grow so that the distance between the nuclei remains equal. In this manner, nuclei are able to compare their relative position and maintain even spacing in all directions. A prerequisite for this mechanism is the anchoring of the astral MT to the nuclear membrane in a polarized fashion, and this function is provided by the MSP-300 ring, which might associate with MT in a klar
Our experiments suggest a major function for MSP-300 both in promoting nuclear spacing (cooperatively with Klar) and in mediating anchoring of the nuclei as well as other organelles to the muscle core myofibrillar domain. The spectrin repeats domain is critical for this latter function, as most of it is eliminated in msp-300Δ3′
. Similarly to other proteins containing the spectrin repeats domain, the multiple (52) spectrin repeats are likely to promote formation of MSP-300 dimers that form elongated filaments capable of connecting between different organelles and the cytoskeleton. In striated muscle, we suggest that such filaments connect between the various organelles and the Z-discs. The association of these filaments with the Z-discs requires the activity of d
-Titin/Sallimus, another large protein that associates with the Z-discs (Zhang et al., 2000
). The function of the N-terminal domain of MSP-300 containing the calponin-homology (CH) actin-binding motifs is still elusive because of the early lethality of the homozygous mutants.
Our analysis provides a mechanistic explanation for the severity of Nesprin-related diseases. We suggest that as in Drosophila
, Nesprin 1 and 2 and a mammalian klar
orthologue (yet to be identified) cooperate to promote myonuclei positioning in vertebrate muscle fibers. Thus, elimination of the KASH domains of both nesprin1
in mutant mice might still leave a putative mammalian Klar orthologue intact, thereby only partially affecting nuclear positioning in the mutant muscles. However, elimination of additional domains of Nesprin described in Zhang et al. (2010)
might abrogate MT organization more profoundly so that other Nesprins are incapable of complementing this situation. In addition, the position of other organelles might be disrupted as observed in msp-300Δ3′
, leading to a more severe phenotype in mice, and possibly also in humans.
A recent study suggested that the amyotrophic lateral sclerosis (ALS)-associated protein VAPB is secreted from motor neurons and promotes the correct positioning of mitochondria in muscle fibers (Han et al., 2012
). It would be interesting to test for a possible functional link between VAPB and MSP-300 activity in promoting mitochondrial positioning in striated muscles.
In summary, our studies provide a mechanistic explanation for the process of myonuclear positioning at distinct developmental stages in striated muscles. These studies may help in the prognosis of the severity of various Nesprin-related diseases in which distinct domains are missing, and provide a basis for future therapeutic approaches.