Biology subjects

Oberhauser, A. F.

Publications and source records attributed to Oberhauser, A. F..

6 recordsLinked to original sources

bioRxiv2023

FARL-11 (STRIP1/2) is Required for Sarcomere and Sarcoplasmic Reticulum Organization in C. elegans

Protein phosphatase 2A (PP2A) functions in a variety of cellular contexts. PP2A can assemble into four different complexes based on the inclusion of different regulatory or targeting subunits. The B regulatory subunit "striatin" forms the STRIPAK complex consisting of striatin, a catalytic subunit (PP2AC), striatin interacting protein 1 (STRIP1), and MOB family member 4 (MOB4). In yeast and C. elegans, STRIP1 is required for formation of the endoplasmic reticulum (ER). Since the sarcoplasmic reticulum (SR) is the highly organized muscle-specific version of ER, we sought to determine the function of the STRIPAK complex in muscle using C. elegans. CASH-1 (striatin) and FARL-11 (STRIP1/2) form a complex in vivo, and each protein is localized to SR. Missense mutations and single amino acid losses in farl-11 and cash-1 each result in similar sarcomere disorganization. A missense mutation in farl-11 shows no detectable FARL-11 protein by immunoblot, disruption of SR organization around M-lines, and altered levels of the SR Ca+2 release channel UNC-68. SummaryProtein phosphatase 2A forms a STRIPAK complex when it includes the targeting B subunit "striatin" and STRIP1. STRIP1 is required for formation of ER. We show that in muscle STRIP1 is required for organization of SR and sarcomeres.

cell biology↗

bioRxiv2022

UNC-45 has a crucial role in maintaining muscle sarcomeres during aging in C. elegans

Summary/AbstractAs people live longer, age-related diseases, like sarcopenia, will become a greater public health concern. We use the model organism C. elegans to better understand the molecular mechanisms behind muscle maintenance. Muscle function is dependent on having properly organized and functioning thick filaments, which are primarily composed of myosin. The myosin head requires the chaperone UNC-45 to initially fold it after translation and is likely used to re-fold back to functionality after thermal or chemical stress induced unfolding. We observe an early onset of sarcopeania when UNC-45 is perturbed during adulthood. We observe that during adult aging, there is a sequential decline of HSP-90, UNC-45, and then myosin. Myosin and UNC-45 protein decline are independent of steady state mRNA levels. Loss of UNC-45 is correlated with an increase in phosphorylation of the protein. By mass spectrometry, S111 was identified as being phosphorylated and this modification may affect binding to HSP-90. A longevity mutant with delayed onset of sarcopenia also shows a delay in the loss of HSP-90, UNC-45, and myosin. We also see a decrease in UNC-45 protein, but not transcript, in an hsp-90 loss of function mutant, suggesting a role for HSP-90 in stabilizing UNC-45. This leads us to propose the model that during aging, a loss of HSP-90 leads to UNC-45 being post translationally modified, such as phosphorylation, and degraded, which then leads to a loss of myosin, and thus muscle mass and function. A better understanding of how myosin and its chaperone proteins are regulated and affected by aging will lead to better preventative care and treatment of sarcopenia and, possibly, the age-related decline of heart muscle function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/494828v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1040254org.highwire.dtl.DTLVardef@34f503org.highwire.dtl.DTLVardef@c1cd67org.highwire.dtl.DTLVardef@1945cf9_HPS_FORMAT_FIGEXP M_FIG C_FIG In young adults, under normal conditions the UCS domain of UNC-45 (shown in green) is bound to the myosin head (in red) and the TPR domain (in yellow) is bound to HSP-90 (in purple). Under stress conditions, HSP-90 detaches from the TPR domain, causing a conformational change in UNC-45 that allows the Central domain (in blue) to bind to the myosin neck (in red) resulting in inhibition of the myosin power stroke while the UCS domain protects/re-folds the myosin head. HSP-90 can then rebind the TPR domain, causing the Central domain to release the myosin neck, allowing movement of the myosin motor. However, aged adults experience a loss of HSP-90 and UNC-45 (which has increased post translational modification with aging). The loss of the Myosin chaperones leads to increased aggregation and degradation of Myosin with age.This loss of Myosin at the thick filament results in decline in muscle mass and function, also known as sarcopenia. Note that only the myosin head and neck are shown for simplicity of illustration.

cell biology↗

bioRxiv2022

Genetic Analysis Suggests a Surface of PAT-4 (ILK) that Interacts with UNC-112 (kindlin)

The transmembrane protein integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a four protein complex (Kindlin, Integrin linked kinase (ILK), PINCH, and parvin). C. elegans muscle provides an excellent model to study integrin adhesion complexes. In C. elegans, UNC-112 (Kindlin) binds to the cytoplasmic tail of PAT-3 ({beta}-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 (ILK) and UNC-112 (Kindlin), we took a genetic approach. First, we isolated mutant PAT-4 proteins that cannot bind to UNC-112. Then, we isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, the mutant PAT-4 protein could localize to attachment structures. Third, overexpression of a mutant PAT-4 protein results in disorganization of the adhesion plaques at muscle cell boundaries, and co-expression of the UNC-112 supressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.

genetics↗

bioRxiv2021

Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity

Proper muscle development and function depends on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination of in vivo C. elegans experiments and in vitro biophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in the in vivo setting and that these conserved regions may be involved in UNC-45 protein stability/ regulation via post translational modifications, protein-protein interactions, or some other unknown mechanism.

molecular biology↗

bioRxiv2020

A Region of UNC-89 (obscurin) Lying Between Two Protein Kinase Domains is a Highly Elastic Spring Required for Proper Sarcomere Assembly

In C. elegans, unc-89 encodes a set of giant multi-domain proteins (up 8,081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, high proline content and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals express normal levels of giant internally deleted UNC-89 proteins, and yet show severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generated in-frame deletion to contain an HA tag at the N-terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake. HighlightsO_LIThe giant muscle proteins UNC-89 and its mammalian homologs have an [~]700 aa non-domain region lying between two protein kinase domains C_LIO_LIBy single molecule force spectroscopy UNC-89 non-domain region is an elastic random coil C_LIO_LINematodes lacking this non-domain region have disorganized sarcomeres and reduced whole animal locomotion C_LIO_LIUNC-89 non-domain region is required for proper assembly of A-bands from thick filaments C_LI

cell biology↗

bioRxiv2020

Mutational analysis of the structure and function of the chaperone domain of UNC-45B

UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. We have previously demonstrated that its UCS domain is responsible for the chaperone-like properties of UNC-45B. In order to better understand the chaperoning function of the UCS domain we engineered mutations designed to: i) disrupt chaperone-client interactions by removing and altering the structure of the putative client-interacting loop and ii) disrupt chaperone-client interactions by changing highly conserved residues in the putative client-binding groove. We tested the effect of these mutations by using a novel combination of complementary biophysical (circular dichroism, intrinsic tryptophan fluorescence, chaperone activity, and SAXS) and in vivo tools (C. elegans sarcomere structure). Removing the client-holding loop had a pronounced effect on the secondary structure, thermal stability, solution conformation and chaperone function of the UCS domain. These results are consistent with previous in vivo findings that this mutation neither rescue the defect in C. elegans sarcomere organization nor bind to myosin. We found that mutating several conserved residues in the client-binding groove do not affect UCS domain secondary structure or structural stability but reduced its chaperoning activity. We found that these groove mutations also significantly altered the structure and organization of the worm sarcomeres. We also tested the effect of R805W, a mutation distant from the client-binding region. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic effect on the structure and organization of the worm sarcomeres. In humans, the R805W mutation segregates with human congenital/infantile cataract, indicating a crucial role of R805 in UCS domain stability and/or client interaction. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin/chaperone interactions in mechanistic detail. Statement of SignificanceThe folding of myosin and the assembly of a functional sarcomere requires the chaperone UNC-45B. The molecular mechanism(s) for how UNC-45B assist in this assembly process or prevent stress-induced aggregation states are presently unknown. Answering this question is a problem at the core of muscle development and function. Here we developed a novel approach that combines biophysical and biological tools to study UNC-45B/myosin interactions in mechanistic detail. Our approach may provide critical insights into the molecular nature of the pathogenesis of many muscle disorders stemming from mutations in sarcomeric proteins including skeletal myopathies and cardiomyopathies, and possibly the age-associated decline in muscle mass and function found in the elderly known as sarcopenia.

biophysics↗