Overexpression of Drosophila NUAK or Constitutively-Active Formin-Like Promotes the Formation of Aberrant Myofibrils

Abstract

Muscle development and maintenance is central to the normal functioning of animals. Muscle tissues exhibit high levels of activity and require the dynamic turnover of proteins. An actomyosin scaffold functions with additional proteins comprising the basic contractile subunit of striated muscle, known as the sarcomere. Drosophila muscles are similar to vertebrate muscles in composition and they share a similar mechanism of development. Drosophila NUAK (NUAK) is the homolog of NUAK1 and NUAK2 in vertebrates. NUAK belongs to the family of AMP-activated protein kinases (AMPKs), a group of proteins with broad and overlapping cellular targets. Here we confirm that NUAK dynamically modulates larval muscle sarcomere size as upregulation of NUAK produces longer sarcomeres, including increased thin filament lengths. Furthermore, NUAK overexpression results in aberrant myofibers above the nuclei plane, upregulation of Formin-like (Frl), and an increase in newly synthesized proteins at sites consistent with actin filament assembly. Expression of constitutively-active Frl also produces aberrant myofibers similar to NUAK overexpression. These results taken together strongly suggest a functional link between NUAK and Frl in myofibril formation in an in vivo setting.

Keywords: Frl; NUAK; actin; sarcomere.

Figure 1.. Sarcomere length and patterning is altered upon muscle-specific NUAK overexpression.

Maximum intensity projections of VL3 and VL4 L3 muscles stained to label Actin (cyan), NUAK (magenta) or nuclei (yellow). (A-A’’) w¹¹¹⁸ (wild type) larval muscle preparation showing the top, or superficial layer in A, and the middle layer in A’. A’’ shows the Z series where nuclei are located on the top surface. (B-B’’) In Mef2>NUAK larval muscle preparations, aberrant muscle fibers are evident in the superficial layer (B). Though the sarcomere pattern is not altered in the middle layer, sarcomeres are longer (B’). B’’ shows aberrant muscle fibers are present above the nuclei. In a given length of VL4 muscle Mef2>NUAK has 19 sarcomeres whereas w¹¹¹⁸ has 27 sarcomeres (compare A’’ and B’’). Scale bar is 40 μm for A-B’. (C-D) Box and whisker plots showing sarcomere length changes in the VL3 and VL4 muscles upon NUAK overexpression. Laser power and gain were adjusted in Mef2>NUAK to avoid any saturation of the signal. N=8 for Mef2>lacZ.NLS and N=15 (C) or N=16 (D) for Mef2>NUAK. Two-tailed student t-test was performed. Mean +/− S.D. (*, p < 0.05; ***, p < 0.005)

Figure 2.. NUAK is enriched in regions corresponding to the thin filament.

(A-D’) w¹¹¹⁸ larval muscle preparation shows localized enrichment of NUAK in wandering L3 (wL3) (A-B’) and early L3 (eL3) stage (C-D’) larva. Actin is labeled in cyan, NUAK in magenta, and nuclei in yellow. Enrichment of NUAK is seen throughout the Z-plane (A’, B’ in wL3 and C’, D’ in eL3). i) and iii) NUAK localizes in a broad sarcomeric pattern that corresponds to the thick filament (white arrowheads) and the Z-disc (white arrows). ii) and iv) Representative confocal images show enrichment of NUAK (white arrows) in regions that overlap with F-actin staining in A and C. (E-F’’) Examples of NUAK enrichment in Mef2>lacZ wL3 (E-E’’) and eL3 muscles (F-F’’). Actin is labeled in cyan, NUAK in magenta, and z-disc (Kettin/Sls) in yellow. E-E’’) NUAK overlaps with Z-disc staining (white arrow) and is enriched at borders of the thin filament (edge of F-actin). F-F’’) In eL3 muscles NUAK accumulates at Z-discs (white arrows) and regions bordering F-actin (yellow arrows). Scale bar in A is 40 μm for A-D and 20 μm for i-iv, E-F’’.

Figure 3.. Coordinate increase of NUAK and Frl in NUAK overexpression muscles.

(A-G) Dissections and immunostainings of L3 muscles with Actin labeled in cyan, NUAK in magenta, and Frl in yellow. (A-B) Frl is expressed at low levels in Mef2>lacZ.NLS (control) muscles (see white line in A’ and corresponding line plot in B). (C-D) In Mef2>NUAK overexpression muscles, the levels of Frl are substantially increased, enriched in nuclei (C’) and partially overlap with Actin (see white line in C’ and corresponding line plot in D where white dashed rectangle marks the Actin-enriched thin filament). (E) In Mef2>lacZ.NLS muscles there is localized accumulation of NUAK and Frl (white dashed box). (F-F’’) This NUAK enrichment colocalizes with Actin and Frl (white arrowheads). (G) In Mef2>NUAK muscles, ring-like NUAK structures (white arrowheads) partially overlap with Frl staining. This is a maximum intensity projection of the superficial layer of a larval muscle where extra myofibers are observed. (H-H’’’) High magnification images of a NUAK ring showing colocalization with Frl and Actin. An actin fiber is seen coming out from the NUAK ring (cyan asterisk in H’’). Scale bar is 40 μm in A, C, E, 10 μm in G, and 2 μm in H.

Figure 4.. NUAK overexpression causes an increase in nascent protein expression.

(A-B’’) L3 larval muscle preparations expressing Mef2>MetRS^L262G-EGFP where EGFP is yellow, Actin is cyan, and newly synthesized proteins are marked by Strep in magenta. Mef2>MetRS^L262G-EGFP L3 larva reared on normal food (negative control) show no ANL labeling (A’’), while ANL-fed larva shows specific labeling (B’’). (C-D’) The change in the signal of ANL labeling (Strep) plotted in C’ and D’ are based on a muscle image from Mef2>MetRS^L262G-EGFP L3 larva fed on normal (C) or ANL food (D). Graphs depicts the drop in ANL labeling (in magenta) in deeper muscle planes. The EGFP and Actin signals are plotted in yellow and cyan for comparison. (E-G) ANL labeling (TAMRA) shows increased signal in L3 larval muscles of Mef2>MetRSL262G-myc, UAS-NUAK (F) in comparison to Mef2>MetRSL262G-myc, UAS-lacZ.NLS (E). Quantitation of the TAMRA signal plotted in the bar and whisker plot shown in G. Two-tailed student t-test. Mean +/− S.D. (**, p < 0.01). N=9 for Mef2>lacZ.NLS and N=11 for Mef2>NUAK. (H) NUAK rings show colocalization with TAMRA (new protein synthesis) and Frl puncta. The NUAK ring and Frl puncta are juxtaposed next to the actin fiber. Scale bar is 40 μm for A-F and 2 μm for H.

Figure 5.. Frl CA produces aberrant myofibrils and ring-like structures.

(A-C’’) L3 muscles stained for F-actin (cyan) or Frl (yellow). (A,B) Unorganized myofibrils are present in muscles VL3 and VL4 (Mef2>Frl CA) (A) or muscle VL1 (5053A>Frl CA) (B). Images are maximum intensity projections. (C-C’’) Single plane images that show Frl is present in punctate structures (C) that resemble rings (white arrowheads in C’) and are often associated with F-actin filaments (C’’). Scale bar is 20 μm for A and B and 5 μm for C-C’.