Functional characterisation of the mitochondrial Hsp70 cochaperone Zim17

The mitochondrial zinc finger protein Zim17 belongs to a newly identified class of cochaperones that maintain the function of Hsp70 proteins in mitochondria and plastides of eukaryotic cells, presumably by preventing the aggregation of their respective chaperone partners. However, while its aggregation-preventive function is well demonstrated in vitro, little is known about the influence of the Zim17 interaction on the chaperone activity of mitochondrial Hsp70s (mtHsp70s) and its concurrent effects in the cellular context. Due to the aggregation-protective character of Zim17, recombinant co-expression with the zinc finger protein allowed the purification of the main yeast mtHsp70 Ssc1 from E.coli cells under native conditions. The purified proteins were used to analyse the influence of Zim17 on the solubility of Ssc1 as well as the character and stability of its binding to the chaperone. Zim17 interacted with Ssc1 as a single molecule but tended to form dimers in the absence of the Hsp70 chaperone. Though the presence of Zim17 improved the solubility of recombinant Ssc1 in E.coli cells, substantial amounts of the Hsp70 chaperone still aggregated, even when Zim17 was expressed in saturated amounts. To study the effects of a loss of Zim17 function in the cellular environment, novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae were generated. Yeast cells carrying these mutations showed a temperature- sensitive growth phenotype and a tendency to develop respiratory deficits. On fermentable growth media, the mutant cells were prone to loose their respiratory competence and were inviable at elevated temperatures. In these cells, a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis was observed. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. The induction of the zim17 mutant phenotype by subjection to a high temperature treatment lead to a strong import defect for Ssc1-dependent matrix-targeted precursor proteins that correlated with a significantly reduced binding of newly imported substrate proteins to Ssc1. Both in vitro and in vivo approaches point to the conclusion that Zim17 is not primarily required for the maintenance of mtHsp70 solubility. Instead, a functional analysis of the chaperone cycles of Ssc1 and Ssq1 shows that Zim17 directly assists the functional interaction of mtHsp70 with substrate proteins in a J-protein cochaperone-dependent manner

Xirp Proteins Mark Injured Skeletal Muscle in Zebrafish

Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does not involve cell proliferation or Pax7+ cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now become accessible to detailed molecular and cellular characterizations

Rapid <i>xirp1</i> transcriptional response to myocellular injury.

<p>Whole-mount <i>in situ</i> hybridizations on 33 hpf embryos which were laser-injured either at 29 hpf (bottom row) or at 32 hpf (middle row) at the level of three different somites (dotted lines). <i>xirp1</i> mRNA transcriptional response occurs within one hour after injury and is no longer detectable at 3.5 hours after injury in WT. There is a lack of <i>xirp1</i> mRNA expression upon laser-induced myocellular injury in <i>xirp1^sa0046</i> mutants. Scale bar: 100 µm.</p

Characterization of the P47 antibody.

<p>(A) Myotendinous junction labeling by the P47 antibody which recognizes Xirp1α/γ is absent in <i>xirp1^sa0046</i> mutant embryos at 24 hpf. This staining was performed upon mild fixation. (B) Clonally expressed Xirp1γ is sensitively detected upon standard fixation conditions by the P47 antibody within somitic muscle tissue. Truncated Xirp1γ^ΔFilCBD-GFP which lacks the Filamin C binding domain including the P47 epitope is not detected by the antibody. Under standard fixation conditions, Xirp1α/γ is not detected at the myotendinous junctions. Green: GFP or Xirp1γ-GFP fusion protein; red: Actin; blue: Xirp1α/γ. All scale bars: 25 µm.</p

Clonal analysis of laser-induced myocellular injuries.

<p>(A–B) Laser-injury and recovery within the same embryo. (A) Left: DIC image superimposed with image of individual muscle cells marked by Tg[<i>βactin:α-actinin-gfp</i>] expression (false colored in yellow). Right: laser-injury was performed by targeting one of the α-Actinin-GFP positive muscle cells. Immediately upon laser-injury, α-Actinin-GFP expression within the damaged cell is diminished (arrowheads). Scale bar: 50 µm. (B) Confocal images of two z-scan planes of an immunohistochemical staining show strong Xirp1 expression within 2.5 hours after laser injury in muscle cells directly adjacent to the targeted cell that was most severely affected by the laser-injury (arrowheads). Pictures on the right are details from the insert (white box on the bottom left picture). Green: <i>βactin</i>:α-Actinin-GFP; red: Actin; blue: Xirp1. Scale bar: 50 µm. (C) Confocal z-scan projection of an immunohistochemical staining 5 hours after laser-induced injury reveals that strong Xirp1 expression correlates with a pattern of Tropomyosin distribution that is different from unaffected regions of the somite. Green: Tropomyosin; red: Actin; blue: Xirp1. Scale bar: 20 µm.</p

Transcriptome analysis for genes regulated by Galanthamine treatment in zebrafish.

<p>Summary table listing those genes that are most strongly upregulated in the acetylcholinesterase inhibitor Galanthamine-induced model of muscle injury. For treatment, zebrafish embryos were continuously incubated with Galanthamine between the end of gastrulation and several days of development. Comparisons of fold changes (FC) at 56 hpf and 72 hpf are shown for those genes showing the strongest expression changes. <i>xirp1</i> is more than three-fold upregulated at both time-points under conditions of myocellular injury.</p

Normal cardiogenesis and myofibrillogenesis in the absence of all Xirps.

<p>(A) Neither cardiac nor skeletal muscle sarcomeric organization of myofibrils is affected in 48 hpf <i>xirp1^sa0046</i> mutants. Green: α-Actinin or Myosin; red: Actin. Red scale bars: 10 µm, white scale bars: 5 µm. (B) Sectioned adult cardiac tissue reveals that neither Xirp2a nor Xirp2b are expressed to compensate for the loss of Xirp1. Also, the <i>xirp1^sa0046</i> mutant lacks all three Xirp1 isoforms. Therefore, Xirps are not required for development or maintenance of cardiac tissue. Green: Xirp1; red: Actin. Scale bar: 10 µm. (C) Complete absence of all Xirps within skeletal muscle prior to 24 hpf in <i>xirp1^sa0046</i>/<i>xirp2a</i>MO mutant/morphants. Localization of Xirp1 is not affected in <i>xirp2a</i> morphants and, conversely, Xirp2a localization is normal in <i>xirp1^sa0046</i> mutants. Scale bar: 50 µm. (D) Complete loss of Xirps in <i>xirp1^sa0046</i>/<i>xirp2a</i>MO mutant/morphants does not impair early cardiogenesis (at 21 hpf) or myofibrillogenesis (at 32 hpf). Green: Xirp1 or Myosin; red: Actin. Red scale bar: 50 µm, white scale bar: 5 µm, yellow scale bar: 20 µm.</p

Efficient Morpholino antisense oligonucleotide-mediated knock-down of <i>xirp2a</i> does not affect myofibrillogenesis.

<p>(A) Phenotypically, <i>xirp2a</i> morphants are indistinguishable from WT embryos at 24 hpf. Scale bar: 250 µm. (B) Efficient gene knock-down of <i>xirp2a</i> is assessed by immunohistochemistry at 24 hpf. Green: Xirp2a; red: Actin. Scale bar: 20 µm. (C) Complete loss of Xirp2a does not affect myofibrillogenesis and correct sarcomeric organization of skeletal muscle as determined by immunohistochemistry using an antibody against the A/I junction epitope of Titin and rhodamine phalloidin to label sarcomeric Actin at 24 hpf. Green: Titin; red: Actin. Scale bar: 5 µm.</p

Expression and localization of Xirps within Galanthamine- and laser-induced myocellular wounds.

<p>(A) Schematic diagram summarizing the temporal order of Xirp expression within somitic muscle and in myocellular wounding assays. (B) Galanthamine (GAL) treatment between 80% epiboly and 2 dpf causes severe disruptions of somitic muscle organization and myofibrillar disarray (red: Actin) in 2 dpf zebrafish embryos. Notably, Xirp1 (green) is strongly expressed and localizes within cells most strongly disrupted by the treatment. These effects are completely reversible within several hours of recovery. Scale bar: 50 µm. (C) Details from inserts indicated in B (green: Xirp1; red: Actin). Scale bar: 10 µm. (D, E) Similarly, laser-induced muscle injury induces ectopic Xirp1 and Xirp2a localization to damaged myofibrils. In comparison, Xirp2b is not yet expressed at 33 hpf. Green: Xirp1, Xirp2a or Xirp2b; red: Actin. Arrows indicate the position of laser-induced injury within somitic tissue. Scale bars: 10 µm. Embryos were injured at 27 hpf (D) or 29 hpf (E) and fixed at 33 hpf (D) or 31.5 hpf (E), respectively.</p

Xirp1 marks wounded skeletal muscle cells prior to <i>de novo</i> cell proliferation.

<p>(A) Laser-induced myocellular injuries in 33 hpf old zebrafish embryos after BrdU pulse labeling (between 24–33 hpf). Embryos were wounded at 31 hpf and left to recover for 2 hours. Xirp1⁺ tissue is devoid of BrdU⁺ proliferative cells. Treatment of laser-induced myocellular injuries with the proliferation inhibitor Aphidicolin (together with BrdU between 24–33 hpf) does not affect expression and localization of Xirp1 within damaged tissue. The efficacy of the Aphidicolin treatment is evident from strongly reduced BrdU labelling. Asterisks indicate the position of laser-induced injury within damaged tissue. Red inserts show Xirp1 localization within damaged tissue. Green: BrDU; red: Actin; blue: Xirp1. (B) Consistent with the lack of proliferating cells within Xirp1⁺ damaged tissue, the distribution of Pax7⁺ external cells is not changed compared to control conditions. Arrowheads mark ectopic Xirp1. Red inserts show Xirp1 localization within damaged tissue. Green: Xirp1; red: Actin; blue: Pax7. All scale bars: 50 µm.</p