Precision in situ cryo-correlative light and electron microscopy of optogenetically-positioned organelles.

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles otherwise positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.

Actin-tropomyosin distribution in non-muscle cells.

The interactions of cytoskeletal actin filaments with myosin family motors are essential for the integrity and function of eukaryotic cells. They support a wide range of force-dependent functions. These include mechano-transduction, directed transcellular transport processes, barrier functions, cytokinesis, and cell migration. Despite the indispensable role of tropomyosins in the generation and maintenance of discrete actomyosin-based structures, the contribution of individual cytoskeletal tropomyosin isoforms to the structural and functional diversification of the actin cytoskeleton remains a work in progress. Here, we review processes that contribute to the dynamic sorting and targeted distribution of tropomyosin isoforms in the formation of discrete actomyosin-based structures in animal cells and their effects on actin-based motility and contractility.

Properties of regenerated mouse extensor digitorum longus muscle following notexin injury.

Muscles of mdx mice are known to be more susceptible to contraction-induced damage than wild-type muscle. However, it is not clear whether this is because of dystrophin deficiency or because of the abnormal branching morphology of dystrophic muscle fibres. This distinction has an important bearing on our traditional understanding of the function of dystrophin as a mechanical stabilizer of the sarcolemma. In this study, we address the question: 'Does dystrophin-positive, regenerated muscle containing branched fibres also show an increased susceptibility to contraction-induced damage?' We produced a model of fibre branching by injecting dystrophin-positive extensor digitorum longus muscles with notexin. The regenerated muscle was examined at 21 days postinjection. Notexin-injected muscle contained 29% branched fibres and was not more susceptible to damage from mild eccentric contractions than contralateral saline-injected control muscle. Regenerated muscles also had greater mass, greater cross-sectional area and lower specific force than control muscles. We conclude that the number of branched fibres in this regenerated muscle is below the threshold needed to increase susceptibility to damage. However, it would serve as an ideal control for muscles of young mdx mice, allowing for clearer differentiation of the effects of dystrophin deficiency from the effects of fibre regeneration and morphology.

The Ski proto-oncogene regulates body composition and suppresses lipogenesis.

OBJECTIVE: The Ski gene regulates skeletal muscle differentiation in vitro and and in vivo. In the c-Ski overexpression mouse model there occurs marked skeletal muscle hypertrophy with decreased adipose tissue mass. In this study, we have investigated the underlying molecular mechanisms responsible for the increased skeletal muscle and decreased adipose tissue mass in the c-Ski mouse. APPROACH: Growth and body composition analysis (tissue weights and dual energy X-ray absorptiometry) coupled with skeletal muscle and white adipose gene expression and metabolic phenotyping in c-Ski mice and wild-type (WT) littermate controls was performed. RESULTS: The growth and body composition studies confirmed the early onset of accelerated body growth, with increased lean mass and decreased fat mass in the c-Ski mice. Gene expression analysis in skeletal muscle from c-Ski mice compared with WT mice showed significant differences in myogenic and lipogenic gene expressions that are consistent with the body composition phenotype. Skeletal muscle of c-Ski mice had significantly repressed Smad1, 4, 7 and myostatin gene expression and elevated myogenin, myocyte enhancer factor 2, insulin-like growth factor-1 receptor and insulin-like growth factor-2 expression. Strikingly, expression of the mRNAs encoding the master lipogenic regulators, sterol-regulatory enhancer binding protein 1c (SREBP1c), and the nuclear receptor liver X-receptor-alpha, and their downstream target genes, SCD-1 and FAS, were suppressed in skeletal muscle of c-Ski mice, as were the expressions of other nuclear receptors involved in adipogenesis and metabolism, such as peroxisome proliferator-activated receptor-gamma, glucocorticoid receptor and retinoic acid receptor-related orphan receptor-alpha. Transfection analysis demonstrated Ski repressed the SREBP1c promoter. Moreover, palmitate oxidation and oxidative enzyme activity was increased in skeletal muscle of c-Ski mice. These results suggest that the Ski phenotype involves attenuated lipogenesis, decreased myostatin signalling, coupled to increased myogenesis and fatty acid oxidation. CONCLUSION: Ski regulates several genetic programs and signalling pathways that regulate skeletal muscle and adipose mass to influence body composition development, suggesting that Ski may have a role in risk for obesity and metabolic disease.

In vivo system for characterizing clonal variation and tissue-specific gene regulatory factors based on function.

The inducibility of stably transfected alpha-cardiac actin genes differs among L cell clones. We examined the ability of muscle-specific factors to induce the expression of the human muscle alpha-cardiac actin gene promoter when stably transfected into mouse fibroblast L cells. This promoter is transcriptionally active in L cells at a low level, 2-5% of that in transfected muscle cells. Upon fusion with muscle cells to form heterokaryons, expression of the transfected alpha-cardiac actin gene promoter can be induced. However, induction is observed with only 10% of transfected L cell clones and the magnitude of this induction varies between 5- and 50-fold. These properties of the transfected L cell appear to be stably inherited. Our results are consistent with the hypothesis that muscle cells contain factors capable of increasing the transcription of the transfected gene, but that differences among L cell clones, possibly in the site of integration in the genome, determine the extent to which the gene can respond. By fusion into heterokaryons, transfectants with responsive genes can be identified. Such clones should prove useful in determining the basis for clonal variation. In addition, they provide an in vivo system for isolating functionally active tissue-specific transcription factors and the genes that encode them.

Plasticity of the differentiated state.

Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.

Identification of a novel slow-muscle-fiber enhancer binding protein, MusTRD1.

The molecular mechanisms which are responsible for restricting skeletal muscle gene expression to specific fiber types, either slow or fast twitch, are unknown. As a first step toward defining the components which direct slow-fiber-specific gene expression, we identified the sequence elements of the human troponin I slow upstream enhancer (USE) that bind muscle nuclear proteins. These include an E-box, a MEF2 element, and two other elements, USE B1 and USE C1. In vivo analysis of a mutation that disrupts USE B1 binding activity suggested that the USE B1 element is essential for high-level expression in slow-twitch muscles. This mutation does not, however, abolish slow-fiber specificity. A similar analysis indicated that the USE C1 element may play only a minor role. We report the cloning of a novel human USE B1 binding protein, MusTRD1 (muscle TFII-I repeat domain-containing protein 1), which is expressed predominantly in skeletal muscle. Significantly, MusTRD1 contains two repeat domains which show remarkable homology to the six repeat domains of the recently cloned transcription factor TFII-I. Furthermore, both TFII-I and MusTRD1 bind to similar but distinct sequences, which happen to conform with the initiator (Inr) consensus sequence. Given the roles of MEF2 and basic helix-loop-helix (bHLH) proteins in muscle gene expression, the similarity of TFII-I and MusTRD1 is intriguing, as TFII-I is believed to coordinate the interaction of MADS-box proteins, bHLH proteins, and the general transcription machinery.

Skeletal muscle of mice with a mutation in slow alpha-tropomyosin is weaker at lower lengths.

Skeletal muscle function was measured in anaesthetised transgenic mice having a mutation in the TPM3 gene (slow alpha-tropomyosin), a similar mutation as found in some patients with nemaline myopathy, and was compared with control muscles. Measurements of isometric and dynamic muscle performance were done with electrical nerve stimulation at physiological temperatures. No muscle weakness was found in the transgenic muscles when performance was measured at muscle optimum length. This was true not only with full activation but also at lower activation levels, indicating that calcium sensitivity was not affected at this length. Also, fatigability was not affected in these conditions. However, isometric force of the muscles with the mutation in TPM3 was lower at lengths below optimum, with more impairment at decreasing length. As the muscles are active over a large range of different muscle lengths during daily activities, this finding may explain, at least in part, the muscle weakness experienced by patients with nemaline myopathy.

The human troponin I slow promoter directs slow fiber-specific expression in transgenic mice.

Troponin I (TnI) is a muscle-specific protein involved in the calcium-mediated contraction of striated muscle. Three TnI isoforms have been identified, each encoded by a separate gene and expressed in specific striated muscles in the adult. The slow isoform gene (TnIs) is transcriptionally regulated during skeletal muscle development such that its expression in the adult is restricted to muscle fibers innervated by a slow nerve. To delineate regions of this gene that are responsive to information imparted by the slow nerve, we generated transgenic mice carrying -4,200 to +12 bp of the human TnIs gene linked to the bacterial chloramphenicol acetyltransferase (CAT) coding region. By Northern blot analysis, we detected transgene transcripts only in muscles containing slow-twitch fibers. CAT histochemical analysis revealed that expression of the transgene is restricted solely to slow-twitch fibers as characterized by type I myosin heavy-chain (MyHC) expression. Using regeneration as a model for neural influenced expression, we show that this gene construct also contains sequences necessary to respond to cues from the central nervous system.

Nerve-responsive troponin I slow promoter does not respond to unloading.

We examined the regulation of the troponin I slow (TnIs) promoter during skeletal muscle unloading-induced protein isoform transition, by using a transgenic mouse line harboring the -4,200 to +12 base pairs region of the human TnIs promoter. Eighteen female transgenic mice ( approximately 30 g body mass) were randomly divided into two groups: weight-bearing (WB) controls (n = 9) and hindlimb unloaded (HU; n = 9). The HU mice were tail suspended for 7 days. Body mass was unchanged in the WB group but was reduced (-6%; P < 0.05) after the HU treatment. Absolute soleus muscle mass (-25%) and soleus mass relative to body mass (-16%) were both lower (P < 0.05) in the HU group compared with the WB mice. Northern blot analyses indicate that 7 days of HU result in a 64% decrease (P < 0.05) in the abundance of endogenous TnIs mRNA (microg/mg muscle) in the mouse soleus. Furthermore, there is a trend for the abundance of the fast troponin I mRNA to be increased (+34%). Analysis of transgenic chloramphenicol acetyltransferase activity in the soleus muscle revealed no difference (P > 0.05) between WB and HU groups. We conclude that additional elements are necessary for the TnIs gene to respond to an unloading-induced, slow-to-fast isoform transition stimulus.

Changes in contractile protein mRNA accumulation in response to spaceflight.

Ten rats were exposed to 9 days of zero gravity aboard the National Aeronautics and Space Administration SLS-1 space mission (June 1991). Levels of fast and slow isoform mRNAs from six contractile protein gene families were quantified in the flight soleus and extensor digitorum longus (EDL) muscles. The gene families studied were myosin light chain-1 (MLC-1), myosin light chain-2 (MLC-2), troponin (Tn) T, TnI, TnC, and tropomyosin. In the EDL muscle there was no change in slow mRNA levels with a general increase in fast mRNA levels from 23 to 232%. Changes in slow mRNA levels were seen in the flight soleus muscle with TnCslow and TnTslow levels increasing slightly, and MLC-1slow a, MLC-1slow b, TnIslow, alpha-Tmslow, and MLC-2slow levels decreasing. All fast mRNA levels increased in the flight soleus muscle from 170 to 1,100%. We can conclude that exposure to zero gravity results in 1) a general increase in fast mRNA levels in both fast and slow muscles and 2) differing directional changes in slow mRNA accumulation in the soleus muscle.

Quantitative analysis of the human alpha-skeletal actin gene in transgenic mice.

Three aspects of the regulation of the human alpha-skeletal actin gene are examined in this study by quantitative analysis of transgenic tissues: level of expression, tissue specificity, and developmental regulation. Previous in vitro and in vivo studies analyzing the 5' end of the gene have indicated that regulation of tissue-specific expression is promoter based. Transgenic mice were produced carrying either a 9.5-kilobase pair (kb) human alpha-skeletal actin gene fragment or a deletion construct with 2.2-kb of 5' sequences of human alpha-skeletal actin linked to the chloramphenicol acetyltransferase reporter gene. We found that the 9.5-kb transgene was capable of expression in adult skeletal muscle at a level equivalent to that of the endogenous gene in a non-transgenic mouse. The deletion construct was also capable of high-level expression. Both transgenes were expressed in a striated muscle-specific manner and were correctly regulated during development. We conclude that these three parameters of regulation of the human alpha-skeletal actin gene are mediated by sequences within the region -2000 to +239 of the promoter.

Overproduction of a Mr 92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells.

We describe a cell line, designated C100, that displays a 100-fold increase in the major regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA; mevalonate:NADP(+) oxido-reductase (CoA-acylating), EC 1.1.1.34]. Immunoprecipitation of [(35)S]methionine-labeled enzyme from C100 microsomal membranes prepared in the presence of the protease inhibitors phenyl-methylsulfonyl fluoride and leupeptin revealed two up regulated proteins: a major band of M(r) 92,000 and a minor band of M(r) 63,000. We conclude that the M(r) 92,000 protein is probably the intact form of HMG-CoA reductase protomer based on the following criteria. (i) It is a highly up regulated microsomal membrane protein that coincides with the increase in HMG-CoA reductase specific activity in this cell line. (ii) It is recognized by a specific HMG-CoA reductase antiserum under a variety of stringencies. (iii) Isolation and solubilization of [(35)S]methionine-labeled C100 microsomal membranes in the absence of protease inhibitors resulted in the disappearance of the M(r) 92,000 protein and the appearance of two proteins of M(r) 52,000 and 38,000. (iv) Analysis of cells labeled for 30 min with [(35)S]methionine, well under the half-life of HMG-CoA reductase, revealed only the M(r) 92,000 protein to be present in total cell extract. (v) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase of M(r) 62,000 [Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982) Proc. Natl. Acad. Sci. USA 79, 1185-1189] can be isolated and appears to be the result of both proteolysis and sample preparation for NaDodSO(4) gel electrophoresis. Analysis of C100 cells labeled with [(35)S]methionine for 24 hr indicates that the predominant steady-state form of the enzyme is the M(r) 92,000, rather than the M(r) 63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship.

Effects of compactin on the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in compactin-resistant C100 and wild-type cells.

A cell line, C100, resistant to 225 microM compactin, has been isolated which overproduces 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase approximately 100-fold compared to the parental cell line [E. Hardeman, H. Jenke and R. Simoni (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1516-1520]. It is demonstrated that the overproduction of HMG-CoA reductase in these cells is the result of increased enzyme synthesis due to elevated levels of translatable mRNA. Furthermore, the apparent molecular weight of the in vitro translation product is 94,000, which agrees with the molecular weight of the in vivo synthesized HMG-CoA reductase protomer in C100 cells. However, a comparison of the Staphylococcus aureus V8 proteolysis patterns between the in vitro and in vivo translation products reveals structural differences which suggests in vivo post-translation modification(s). It is also demonstrated unequivocally, by comparing proteolytic cleavage patterns and pulse-chase experiments, that the previously reported 63,000-, 52,000-, and 38,000-Da polypeptides recognized by HMG-CoA reductase antiserum derive from the 94,000-Da protomer as a result of nonphysiological proteolysis. Finally, the types of regulatory mechanisms involved in both the induction and repression of the enzyme in the presence or absence of compactin were determined. Four biochemical parameters of HMG-CoA reductase were examined in variant and parental cells grown in the presence and absence of compactin: enzymatic activity, degradation rate, synthesis rate, and concentration of translatable mRNA. These studies revealed that changes in cellular HMG-CoA reductase content are a function of concurrent changes in the rates of enzyme degradation and synthesis. Changes in enzyme synthesis are due to alterations in the level of translatable mRNA.

Multiple regions of the human cardiac actin gene are necessary for maturation-based expression in striated muscle.

In order to elucidate mechanisms involved in striated muscle contractile protein isoform expression, we have defined regulatory elements in the cardiac actin gene necessary for postnatal expression at the level of transcript accumulation in the heart and hindlimb muscles of transgenic mice. During this developmental period in the rodent, cardiac actin expression essentially remains constant in the heart, but declines significantly in skeletal muscle. We determined that a 13-kilobase human cardiac actin gene fragment contains sufficient information to direct this maturation-based developmental expression, as well as striated muscle-specific and high level expression. We localized an element responsible for maturation-based down-regulation in the 3' flank of the gene between approximately 950 and 2120 base pairs downstream of the polyadenylation site. Furthermore, we determined that -800 base pairs of 5'-flanking DNA, which contains multiple MyoD1 binding sites, as well as serum response element and AP1 binding sites, can account for striated muscle-specific expression, but not high level expression. Findings indicate that sequence(s) responsible for high level expression of the gene must be located within the body of the gene. We conclude that the human cardiac actin gene contains distinct sequences which confer developmental, tissue-specific, and high level expression.

The pattern of actin expression in human fibroblast x mouse muscle heterokaryons suggests that human muscle regulatory factors are produced.

The expression of previously dormant human muscle genes encoding two major components of the contractile apparatus was activated in multinucleated heterokaryons formed by the fusion of mouse muscle cells and human fibroblasts. The accumulation of human and mouse alpha-cardiac and alpha-skeletal actin transcripts was compared by Northern blot, slot blot, and S1 nuclease assays. The pattern of human transcript accumulation in heterokaryons was quite distinct from that in the mouse muscle cells that induced it, and strikingly similar in time course and relative amounts to that in human primary muscle cultures. In addition, the usual decline in the level of mouse alpha-cardiac actin transcripts was not observed; instead, after fusion with human fibroblasts the levels increased. Our findings suggest that the activated human nuclei in heterokaryons produce their own muscle regulatory factors that alter the expression of mouse muscle genes and direct the expression of the human muscle phenotype.

Alpha-skeletal actin induces a subset of muscle genes independently of muscle differentiation and withdrawal from the cell cycle.

Muscle differentiation is characterized by the induction of genes encoding contractile structural proteins and the repression of nonmuscle isoforms from these gene families. We have examined the importance of this regulated order of gene expression by expressing the two sarcomeric muscle actins characteristic of the differentiated state, i.e. alpha-skeletal and alpha-cardiac actin, in C2 mouse myoblasts. Precocious accumulation of transcripts and proteins for a group of differentiation-specific genes was elicited by alpha-skeletal actin only: four muscle tropomyosins, two muscle actins, desmin and MyoD. The nonmuscle isoforms of tropomyosin and actin characteristic of the undifferentiated state continued to be expressed, and no myosin heavy or light chain or troponin transcripts characteristic of muscle differentiation were induced. Stable transfectants displayed a substantial reduction in cell surface area and in the levels of nonmuscle tropomyosins and beta-actin, consistent with a relationship between the composition of the actin cytoskeleton and cell surface area. The transfectants displayed normal cell cycle progression. We propose that alpha-skeletal actin can activate a regulatory pathway linking a subset of muscle genes that operates independently of normal differentiation and withdrawal from the cell cycle.

Reappearance of the minor alpha-sarcomeric actins in postnatal muscle.

The postnatal expression profiles of alpha-sarcomeric actin transcripts and protein are quantified in mouse striated muscles from birth to postnatal day 56 by Northern and Western blot analyses. alpha-Cardiac actin (alpha-CA) transcripts transiently increase between 12 and 21 days after birth in the quadriceps muscle, reaching approximately 90% that found in the adult mouse heart. Although alpha-CA is the alpha-sarcomeric actin isoform expressed in the immature fiber, the expression profiles of other contractile protein isoforms indicate that this postnatal period is not reflective of an immature phenotype. alpha-Skeletal actin (alpha-SA) transcripts accumulate to approximately 32% of the total alpha-sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a simultaneous reappearance of alpha-CA and alpha-SA in postnatal skeletal and heart muscles, respectively, and 2) the contractile protein gene expression profile characteristic of adult skeletal muscle is not achieved until after 42 days postnatal in the mouse. We propose there is a previously uncharacterized period of postnatal striated muscle maturation marked by the reappearance of the minor alpha-sarcomeric actins.

Isolation and characterization of cells resistant to ML236B (compactin) with increased levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

ML236B is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (EC 1.1.1.34), the major regulatory enzyme in cholesterol biosynthesis. This compound inhibits cell growth when present in the culture medium of CHO-K1 cells at a concentration as low as 0.1 micrograms/ml. Addition of the product of the HMG-CoA reductase reaction, mevalonate, to the culture medium prevents the cytotoxic effects of ML236B at a concentration of inhibitor as high as 50 micrograms/ml. Using a stepwise selection procedure, we have obtained two variant cell lines which are resistant to the presence of 8 micrograms/ml of ML236B in the culture medium. The rates of cholesterol synthesis and the cholesterol levels in the variant cell lines, grown in the presence of ML236B, are similar to those of the parental CHO-K1 cell line grown in the absence of inhibitor. Assays of HMG-CoA reductase activity from extracts of variant cells, grown in the presence of inhibitor, reveal that the variant cell lines have an approximately 40-fold higher HMG-CoA reductase activity than does the parental CHO-K1 cell line grown in the absence of inhibitor. However, when the variant cell lines are grown without ML236B in the culture medium, the HMG-CoA reductase activity returns to the parental CHO-K1 level within 5 days, but the resistant phenotype is stable for up to 9 months. We conclude that the variant cell lines are unable to overcome the cytotoxic effects of ML236B by a mechanism which leads to overaccumulation of HMG-CoA reductase which in turn permits normal mevalonate metabolism and cholesterol synthesis to take place.