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CeAha1 and CeHop are novel modifiers of unc-45 function. (A) Age-synchronized (L1) RW1596 animals, expressing MYO-3:GFP, were grown at 15°C, collected on day 2 of adulthood, stained with specific antibodies to CeAha1 or CeHop, and imaged by confocal microscopy. Scale bar is 10 μm. (B) Confocal images of body wall muscle. Age-synchronized wild-type, unc-45(e286) or unc-45(m94) animals were grown at 25°C, collected on day 2 of adulthood and stained with anti-MYO-3 antibodies. Scale bar is 10 μm. (C) Confocal images of body-wall muscle. Age-synchronized wild-type, unc-45(e286) or unc-45(m94) animals were grown at 15°C, transferred to plates containing control, CeAha1 (C01G10.8) or CeHop (sti-1) RNAi-expressing bacteria, collected on day 2 of adulthood and stained with anti-MYO-3 antibodies. Scale bar is 10 μm.
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Proteome stability is central to cellular function and the lifespan of an organism. This is apparent in muscle cells, where incorrect folding and assembly of the sarcomere contributes to disease and aging. Apart from the myosin-assembly factor UNC-45, the complete network of chaperones involved in assembly and maintenance of muscle tissue is curren...
Citations
... Thus, there would be a mechanism that makes condensates more likely in the body-wall muscle in worms compared to Neuro2a cells. Proteome stability and proteostasis maintenance are widely known to be crucial for the cellular function and lifespan of organisms, and it is important for the maintenance of muscle cells and neurons 42,74 . These findings suggest that misfolded protein oligomers such as C220/TDP25 and C233 and their condensates composed of these oligomers would be toxic and make damage the body-wall muscles. ...
Carboxy terminal fragments (CTFs) of TDP-43 contain an intrinsically disordered region (IDR) and form cytoplasmic condensates containing amyloid fibrils. Such condensates are toxic and associated with pathogenicity in amyotrophic lateral sclerosis. However, the molecular details of how the domain of TDP-43 CTFs leads to condensation and cytotoxicity remain elusive. Here, we show that truncated RNA/DNA-recognition motif (RRM) at the N-terminus of TDP-43 CTFs leads to the structural transition of the IDR, whereas the IDR itself of TDP-43 CTFs is difficult to assemble even if they are proximate intermolecularly. Hetero-oligomers of TDP-43 CTFs that have recruited other proteins are more toxic than homo-oligomers, implicating loss-of-function of the endogenous proteins by such oligomers is associated with cytotoxicity. Furthermore, such toxicity of TDP-43 CTFs was cell-nonautonomously affected in the nematodes. Therefore, misfolding and oligomeric characteristics of the truncated RRM at the N-terminus of TDP-43 CTFs define their condensation properties and toxicity.
... Chaperones such as Hsp70 and Hsp90 recognize exposed hydrophobic regions on misfolded proteins. They maintain these proteins in a foldable state until proper folding is achieved through ATP hydrolysis and cochaperone action (Frumkin et al., 2014;Saibil, 2021). Notably, chaperones also direct misfolded proteins toward degradation. ...
Alzhеimеr's disеasе (AD) is characterized by thе accumulation of misfoldеd amyloid-bеta and tau protеins. Autophagy acts as a protеostasis procеss to rеmovе protеin clumps, although it progrеssivеly wеakеns with aging and AD, thus facilitating thе accumulation of toxic protеins and causing nеurodеgеnеration. This rеviеw еxaminеs thе impact of impairеd autophagy on thе progrеssion of AD disеasе pathology. Undеr normal circumstancеs, autophagy rеmovеs abnormal protеins and damagеd organеllеs, but any dysfunction in this procеss can lеad to thе еxacеrbation of amyloid and tau pathology, particularly in AD. Thеrе is incrеasing attеntion to thеrapеutic tactics to rеvitalizе autophagy, including rеducеd caloric intakе, autophagy-stimulating drugs, and gеnеtic thеrapy. Howеvеr, thе translation of thеsе stratеgiеs into clinical practicе facеs sеvеral hurdlеs. In summary, this rеviеw intеgratеs thе undеrstanding of thе intricatе rolе of autophagy dysfunction in Alzhеimеr's disеasе progrеssion and rеinforcеs thе promising prospеcts of autophagy as a bеnеficial targеt for modifying thе coursе of Alzhеimеr's disеasе trеatmеnt.
... Hsp90a1 is crucial for myofibril organization in skeletal muscle development, whereas Hsp90a2 has no effect on muscle development [46]. Coherent with observations in vertebrates, the only cytosolic Hsp90 isoform in the invertebrate C. elegans (HSP-90) is crucial for myosin folding and muscle development, as RNAi-mediated knockdown leads to disrupted myosin filaments and motility defects [47,48]. Mammals such as mice, however, appear to require higher threshold levels of Hsp90 to promote stress adaptation and survival of the organism compared to yeast. ...
Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone that assists in the maturation of many client proteins involved in cellular signal transduction. As a regulator of cellular signaling processes, it is vital for the maintenance of cellular proteostasis and adaptation to environmental stresses. Emerging research shows that Hsp90 function in an organism goes well beyond intracellular proteostasis. In metazoans, Hsp90, as an environmentally responsive chaperone, is involved in inter-tissue stress signaling responses that coordinate and safeguard cell nonautonomous proteostasis and organismal health. In this way, Hsp90 has the capacity to influence evolution and aging, and effect behavioral responses to facilitate tissue-defense systems that ensure organismal survival. In this review, I summarize the literature on the organismal roles of Hsp90 uncovered in multicellular organisms, from plants to invertebrates and mammals.
... While UCS proteins are key in this regard, their interplay with many of the other chaperones and activities for protein turnover is still poorly understood. Screens have identified a number of other chaperones required for muscle integrity in C. elegans, including CeHop, CeAha1 and Cep23 [81]. Enabling Hsp70/Hsp90, their accessory components and the systems for protein turnover to establish and maintain the intricate myosin-actin interplay clearly presents a major challenge for the cellular chaperone machinery. ...
The folding of the myosin head often requires a UCS (Unc45, Cro1, She4) domain-containing chaperone. Worms, flies, and fungi have just a single UCS protein. Vertebrates have two; one (Unc45A) which functions primarily in non-muscle cells and another (Unc45B) that is essential for establishing and maintaining the contractile apparatus of cardiac and skeletal muscles. The domain structure of these proteins suggests that the UCS function evolved before animals and fungi diverged from a common ancestor more than a billion years ago. UCS proteins of metazoans and apicomplexan parasites possess a tetratricopeptide repeat (TPR), a domain for direct binding of the Hsp70/Hsp90 chaperones. This, however, is absent in the UCS proteins of fungi and largely nonessential for the UCS protein function in Caenorhabditis elegans and zebrafish. The latter part of this review focusses on the TPR-deficient UCS proteins of fungi. While these are reasonably well studied in yeasts, there is little precise information as to how they might engage in interactions with the Hsp70/Hsp90 chaperones or might assist in myosin operations during the hyphal growth of filamentous fungi.
... Similarly, knockdown of Aha1 (ahsa-1) expression in Caenorhabditis elegans had no apparent phenotype under normal condition. However, under stress conditions from unc-45 temperature-sensitive mutation and Hsp90 deficiency, Aha1 (ahsa-1) was found to be required for muscle homeostasis and integrity (Frumkin et al., 2014). A recent study demonstrated that overexpression of Aha1 promotes tau pathogenesis in aged mice (Criado-Marrero et al., 2021). ...
... Given that aha1a and aha1b are cochaperones of Hsp90 required for muscle development in C. elegans and zebrafish embryos (Du et al., 2008;Frumkin et al., 2014), we tested whether loss of aha1a and aha1b could trigger an upregulation of heat shock protein gene expression. hsp90α1 and hsp90α2 gene expression was analyzed during myogenesis in aha1a +17 ;aha1b − 4 double mutant embryos by whole mount in situ hybridization and qRT-PCR. ...
... Biochemical analysis showed that Aha1 and Hch1 were essential cochaperones of Hsp90 and played a vital role in Hsp90 cycle and client protein activation (Lotz et al., 2003;Panaretou et al., 2002). Genetic analysis in C. elegans revealed similar findings with respect to Aha1 function in muscle development and animal mobility (Frumkin et al., 2014). Frumkin and colleagues reported that knockdown of Aha1 (ahsa-1) expression in Caenorhabditis elegans had no apparent phenotype under normal condition. ...
Activator of heat shock protein 90 (hsp90) ATPase (Aha1) is a Hsp90 co-chaperone required for Hsp90 ATPase activation. Aha1 is essential for yeast survival and muscle development in C. elegans under elevated temperature and hsp90-deficeiency induced stress conditions. The roles of Aha1 in vertebrates are poorly understood. Here, we characterized the expression and function of Aha1 in zebrafish. We showed that zebrafish genome contains two aha1 genes, aha1a and aha1b, that show distinct patterns of expression during development. Under normal the physiological condition, aha1a is primarily expressed in skeletal muscle cells of zebrafish embryos, while aha1b is strongly expressed in the head region. aha1a and aha1b expression increased dramatically in response to heat shock induced stress. In addition, Aha1a-GFP fusion protein exhibited a dynamic translocation in muscle cells in response to heat shock. Moreover, upregulation of aha1 expression was also observed in hsp90a1 knockdown embryos that showed a muscle defect. Genetic studies demonstrated that knockout of aha1a, aha1b or both had no detectable effect on embryonic development, survival, and growth in zebrafish. The aha1a and aha1b mutant embryos showed normal muscle development and stress response in response to heat shock. Single or double aha1a and aha1b mutants could grow into normal reproductive adults with normal skeletal muscle structure and morphology compared with wild type control. Together, data from these studies indicate that Aha1a and Aha1b are involved in stress response. However, they are dispensable in zebrafish embryonic development, growth, and survival.
... Muscle chaperones, most of which are hlh-1-dependent, are required for muscle proteostasis (Bar-Lavan et al., 2016b;Nisaa and Ben-Zvi, 2021;Shemesh et al., 2021). Disrupting the expression of a single muscle chaperone by knockdown or overexpression often affects muscle proteome folding and function and, for some chaperones, can even disrupt muscle differentiation (Landsverk et al., 2007;Frumkin et al., 2014;Papsdorf et al., 2014;Bar-Lavan et al., 2016b;Echeverria et al., 2016;Nisaa and Ben-Zvi, 2021;Tiago et al., 2021). For example, DNJ-24, UNC-23, HSP-90, and STI-1 are localized to the sarcomere, and when any are disrupted, myosin is disorganized (Meissner et al., 2011;Frumkin et al., 2014;Papsdorf et al., 2014;Rahmani et al., 2015;Bar-Lavan et al., 2016b). ...
... Disrupting the expression of a single muscle chaperone by knockdown or overexpression often affects muscle proteome folding and function and, for some chaperones, can even disrupt muscle differentiation (Landsverk et al., 2007;Frumkin et al., 2014;Papsdorf et al., 2014;Bar-Lavan et al., 2016b;Echeverria et al., 2016;Nisaa and Ben-Zvi, 2021;Tiago et al., 2021). For example, DNJ-24, UNC-23, HSP-90, and STI-1 are localized to the sarcomere, and when any are disrupted, myosin is disorganized (Meissner et al., 2011;Frumkin et al., 2014;Papsdorf et al., 2014;Rahmani et al., 2015;Bar-Lavan et al., 2016b). Indeed, when the functional association of chaperones that are upregulated in human skeletal muscle (and their C. elegans homologs) was examined, most were required for muscle function, and more than half were causal or associated with muscle diseases (Shemesh et al., 2021). ...
Muscle proteostasis is shaped by the myogenic transcription factor MyoD which regulates the expression of chaperones during muscle differentiation. Whether MyoD can also modulate chaperone expression in terminally differentiated muscle cells remains open. Here we utilized a temperature-sensitive (ts) conditional knockdown nonsense mutation in MyoD ortholog in C. elegans, HLH-1, to ask whether MyoD plays a role in maintaining muscle proteostasis post myogenesis. We showed that hlh-1 is expressed during larval development and that hlh-1 knockdown at the first, second, or third larval stages resulted in severe defects in motility and muscle organization. Motility defects and myofilament organization were rescued when the clearance of hlh-1(ts) mRNA was inhibited, and hlh-1 mRNA levels were restored. Moreover, hlh-1 knockdown modulated the expression of chaperones with putative HLH-1 binding sites in their promoters, supporting HLH-1 role in muscle maintenance during larval development. Finally, mild disruption of hlh-1 expression during development resulted in earlier dysregulation of muscle maintenance and function during adulthood. We propose that the differentiation transcription factor, HLH-1, contributes to muscle maintenance and regulates cell-specific chaperone expression post differentiation. HLH-1 may thus impact muscle proteostasis and potentially the onset and manifestation of sarcopenia.
... Proteome stability and proteostasis maintenance are widely known to be crucial for the cellular function and lifespan of organisms, and it has been shown to be important for the maintenance of muscle cells and neurons. (Ben-Zvi et al., 2009;Frumkin et al, 2014). These findings suggest that misfolded protein oligomers such as C220/TDP25 and C233 and their condensates composed of these oligomers would be toxic and make damage the body-wall muscles. ...
Carboxy terminal fragments (CTFs) of TDP-43 contain an intrinsically disordered region (IDR) and form cytoplasmic condensates containing amyloid fibrils. Such condensates are toxic and associated with pathogenicity in several neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. However, the molecular details of how the domain of TDP-43 CTFs leads to condensation and cytotoxicity remain elusive. Here, we show that truncated RNA/DNA-recognition motif (RRM) at the N-terminus of TDP-43 CTFs is assembled and leads to the structural transition of the IDR, whereas the IDR itself of TDP-43 CTFs is difficult to assemble even if they are proximate intermolecularly. Hetero-oligomers of TDP-43 CTFs that have recruited other proteins that are essential for proteostasis into low mobile condensates are more toxic than homo-oligomers inside highly mobile condensates, implicating loss-of-function of the endogenous proteins by such oligomers, not necessarily the condensates, is associated with cytotoxicity. Furthermore, such toxicity of TDP-43 CTFs was cell-nonautonomously affected in the nematodes. We speculate that the misfolding and oligomeric characteristics of the truncated RRM at the N-terminus of TDP-43 CTFs define their condensation properties and toxicity by implanting and transmitting structures with toxic properties of the truncated RRM into the IDR.
... Knocking down myrf-2 in BWM reduced the gaps and aggregates in the aged sarcomeres (Fig 7B and C). The disruption of sarcomeres is closely related to the decreasing motility of animals (Frumkin et al, 2014). Consistent with its effect on sarcomere structure, BWM-specific RNAi against myrf-2 also improved the motility of aged worms (Fig 7D and E and Movie EV1). ...
Ageing is a complex process with common and distinct features across tissues. Unveiling the underlying processes driving ageing in individual tissues is indispensable to decipher the mechanisms of organismal longevity. Caenorhabditis elegans is a well-established model organism that has spearheaded ageing research with the discovery of numerous genetic pathways controlling its lifespan. However, it remains challenging to dissect the ageing of worm tissues due to the limited description of tissue pathology and access to tissue-specific molecular changes during ageing. In this study, we isolated cells from five major tissues in young and old worms and profiled the age-induced transcriptomic changes within these tissues. We observed a striking diversity of ageing across tissues and identified different sets of longevity regulators therein. In addition, we found novel tissue-specific factors, including irx-1 and myrf-2, which control the integrity of the intestinal barrier and sarcomere structure during ageing respectively. This study demonstrates the complexity of ageing across worm tissues and highlights the power of tissue-specific transcriptomic profiling during ageing, which can serve as a resource to the field.
... A genetic interaction screen using RNAi can, therefore, reveal the impact of down-regulating a set of genes or most C. elegans coding genes using RNAi libraries 24 . In such a screen, hits that impact the behavior of the mutant of interest but not the wild type strain are potential modifiers of the phenotype being monitored 25 . Here, we apply a combination of mutations and RNAi screening to systematically map tissue-specific chaperone interactions in C. elegans. ...
... Mutations in UNC-45 have been shown to induce myosin disorganization and severe motility defects in C. elegans 31,36 . UNC-45 tandem modules assemble into a multi-site docking platform 37 that enforces collaboration between UNC-45, HSP-90 and HSP-1 and likely other chaperones and co-chaperones in myosin filament assembly 25,36,37,38 . To confirm known UNC-45 interactions and identify novel genetic interactions in muscle proteostasis, we established a strategy using C. elegans temperature-sensitive unc-45 mutations as a sensitized genetic background for tissue-specific chaperone interaction screening 19,25 . ...
... UNC-45 tandem modules assemble into a multi-site docking platform 37 that enforces collaboration between UNC-45, HSP-90 and HSP-1 and likely other chaperones and co-chaperones in myosin filament assembly 25,36,37,38 . To confirm known UNC-45 interactions and identify novel genetic interactions in muscle proteostasis, we established a strategy using C. elegans temperature-sensitive unc-45 mutations as a sensitized genetic background for tissue-specific chaperone interaction screening 19,25 . ...
Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.
... Hence, we propose that ectopic EFF-1 expression in host cells, such as body wall muscles (BWMs), could retarget VSV∆G-AFF-1 into these cells. C. elegans has 95 mononucleated rhomboid BWMs, arranged as staggered pairs and bundled in four quadrants that run along the worm's body [41][42][43]. While most vertebrates and invertebrates have syncytial striated muscles composed of long multinucleated myofibers, in C. elegans and other nematodes the BWMs have not been Most adult animals, including myo-3p::mCherry (+) and (-), were wild-type-like (Table S1, Figures 4A-4B and Movie S1), while about 10% of animals were both myo-3p::mCherry(+) and had an uncoordinated and dumpy (Unc+Dpy) phenotype (Table S1, Figures 4C-4D and Movie S1). ...
A hallmark of muscle development is that myoblasts fuse to form myofibers. However, smooth muscles and cardiomyocytes do not generally fuse. In C. elegans , the body wall muscles (BWMs), the physiological equivalents of skeletal muscles, are mononuclear. Here, to determine what would be the consequences of fusing BWMs, we express the cell-cell fusogen EFF-1 in these cells. We find that EFF-1 induces paralysis and dumpy phenotypes. To determine whether EFF-1-induced muscle fusion results in these pathologies we injected viruses pseudotyped with AFF-1, a paralog of EFF-1, into the pseudocoelom of C. elegans . When these engineered viruses encounter cells expressing EFF-1 or AFF-1 they are able to infect them as revealed by GFP expression from the viral genome. We find that AFF-1 viruses can fuse to EFF-1-expressing muscles revealing multinucleated fibers that cause paralysis and abnormal muscle morphogenesis. Thus, aberrant fusion of otherwise non-syncytial muscle cells may lead to pathological conditions.
Graphical abstract
Significance statement
Most cells are individual units that do not mix their cytoplasms. However, some cells fuse to become multinucleated in placenta, bones and muscles. In most animals, muscles are formed by myofibers that originate by cell-cell fusion. In contrast, in C. elegans the body wall muscles are mononucleated cells that mediate worm-like movement. EFF-1 and AFF-1 fusogens mediate physiological cell fusion in C. elegans . By ectopically expressing EFF-1 in body wall muscles we induce their fusion resulting in behavioral and morphological deleterious effects, revealing possible causes of congenital myopathies in humans. Using AFF-1-coated pseudoviruses we infect EFF-1-expressing muscle cells retargeting viral infection into these cells. We suggest that virus retargeting can be utilized to study myogenesis, neuronal regeneration, gamete fusion and screens for new fusogens in different organisms. In addition, our virus retargeting system can be used in gene-therapy, viral-based oncolysis and to study viral-host interactions.
