SALS-WH2
SALS-Pro-WH2
The morphology and dynamics of sarcomeric actin filaments are essential for proper muscle development and function. However, it is not completely understood how sarcomeric actin filaments are stereotyped in length and dynamics.
Recently, Bai et al.
(1)identified a WH2-domain-containing protein, SALS (sarcomere length short) in Drosophila as an important regulator of the assembly of sarcomeric actin structures. Disruption of sals by RNAi results in lethality at the embryonic stages. This phenotype results from muscle defects caused by the improper organisation of sarcomeric actin filaments in SALS mutant embryos. Further loss-, and gain-of-function studies in Drosophila primary muscle cells derived from myoblasts indicate that SALS influences sarcomere length by promoting actin filament assembly at the pointed ends (Figure 1). SALS is also proposed to antagonise with the pointed end-binding protein, tropomodulin. However, the exact role of SALS in muscle development has not been revealed.
(1) Bai J, Hartwig JH, Perrimon N. SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth. Dev Cell.2007 Dec;13(6):828-42.
Mónika Ágnes Tóth 1 , József Mihály 2 , Ede Migh 2 , Miklós Nyitrai 1 and Beáta Bugyi 1
1 University of Pécs, Medical School, Department of Biophysics, Pécs Szigeti str 12. H-7624
2 Biological Research Centre, Centre of Excellence of the European Union, Institute of Genetics, Laboratory of Actin Cytoskeleton Regulation, Szeged, Temesvári bvd. 62. H-6726 e-mail: beata.bugyi@aok.pte.hu
Figure 1. Confocal micrographs of myofibrils from Drosophila embryos at 20–22 hr AEL.
(1) Sarcomere: Z-Z region (red)
OTKA-PD83648 (BB) NKTH-REG-DD-09-1-2009-0009-Tirfm_09 (BB) Bolyai János Fellowship, Hungarian Academy of Sciences (BB) SROP-4.2.2. B-10/1-2010-0029
SROP-4.2.1. B-10/2/KONV-2010-0002 SROP-4.2.2-08/1-2008-0011
Figure 7. Prepolymerised actin (containing 5% pyrenyl labelled actin) were incubated with SALS-WH2 overnight, and the fluorescene emission of the samples were measured.
Note: the breaking point reflects the value of the critical concentration (c
c); the steady-state amount of free actin monomers established by filament ends dynamics.
barbed end: cc ≈ 70 nM pointed end: cc ≈ 600 nM
barbed end + pointed end ≈ 100 nM
PELLET: FILAMENT
SUPERNATANT: MONOMER SALS-WH2
ACTIN
SALS-WH2
ACTIN
SALS-WH2
Figure 3. Prepolymerised actin filaments (2.5 mM) were incubated overnight with SALS-WH2.
Samples were ultracentrifuged (UC), the pellets and supernatants were separated and processed for SDS-page analysis. Coomassie blue stained gels of pellets and supernatants (left panel). Actin content of pellets and supernatants as the function of SALS-WH2 concentration (middle panel). Some samples were stained with fluorescent phalloidin (1 : 1 molar ratio to actin) and processed for microscopy observation before UC (right panel).
Note: the actin content of the pellets (filaments) decreases, in paralell the actin accumulates in the supernatatns (monomer).
Figure 5. To study nucleation and barbed end assembly SALS-WH2 at different concentrations was added to pyrenyl labelled actin monomers simultaneously with the initiation of actin polymerisation (100 mM KCl and 2 mM MgCl2) and the fluorescence intensity of the samples was measured as a function of time.
Note: the fluorescence intensity of pyrene-actin increases upon monomer-filament transition reflecting the kintetics of actin assembly.
Figure 2. Domain organisation of SALS (upper panel). Comparative sequence analysis of SALS’s WH2 domains (lower panel). Pro: proline-rich region, WH2: WH2 domain, NLS: predicted nuclear localisation signal region
Figure 6. Steady-state anisotropy of IAEDANS labeled ATP actin monomers (G) as the function of SALS concentration.
Note: anisotropy increase reflects complex formation.
Loss of function: SHORTER SARCOMERES
Gain of function: LONGER SARCOMERES
To dissect the mechanism by which SALS contribute to the establishment of sarcomeric actin structures first we investigate the interaction of SALS with actin using biochemical and biophysical approaches. We address the following questions:
The biochemical properties of SALS-WH2 revealed by our study suggest the opposite functional behavior than that is observed in vivo.To reveal which factors are necessary for SALS’ biological function we address the following questions:
barbed pointed
+ SALS WH2
𝒂𝒔𝒔𝒆𝒎𝒃𝒍𝒚 𝒓𝒂𝒕𝒆 = 𝒌 + 𝑭 𝑮
𝒅𝒊𝒔𝒂𝒔𝒔𝒆𝒎𝒃𝒍𝒚 𝒓𝒂𝒕𝒆 = 𝒌 − 𝑭 𝑮 ↓ 𝑭 ↑
WT
- SALS
actin tropomodulin
WT
+ SALS
actin titin homolog
0 1 2 3 4 5 6
0.140 0.145 0.150 0.155 0.160 0.165 0.170 0.175
steady-s tate anisot ropy
[SALS-WH2],
mM
1 mM IAEDANS G-actin Mg2+
Ca2+
0.0 2.5 5.0 7.5 10.0 12.5 15.0 0
200 400 600 800 1000 1200 1400
act in, a. u.
[SALS-WH2], m M
[actin]
total = 2.5 mM
pellet: filament supernatant: monomer
Figure 4. To study barbed end disassembly, prepolymerised pyrenyl labelled actin filaments (F) were diluted to 20 nM in the absence or presence of SALS-WH2, as indicated (letf panel). Tropomyosin (skTm)- bound actin filaments were diluted to 20 nM in the presence of 4.5 mM skTm and in the absence or presence of SALS-WH2, as indicated (right panel).
The fluorescence intensity of the samples was measured as a function of time.
Note: the fluorescence intensity of pyrene-actin decreases upon filament-monomer transition reflecting the kintetics of actin disassembly.
0 500 1000 1500
0 2000 4000 6000 8000 10000
py reny l fl uores cenc e, a.u.
[actin], nM [SALS-WH2],
mM
0 2
barbed end pointed end
+ SAL S- WH2
0 200 400 600 800 1000
0.6 0.7 0.8 0.9 1.0 1.1
relative pyrenyl fluoresence
time, s
1mM F-actin
20 nM (50 % PA)
[SALS-WH2], mM 0
0.1 0.5 1.5 6
0 200 400 600 800 1000 0.6
0.7 0.8 0.9 1.0 1.1
relat ive pyr eny l f luore sc enc e
time, s
1mM F-actin + 4.5 mM skTm
20 nM + 4.5 mM skTm (50 % PA)
[SALS-WH2], mM 0
0.1 2 6
