Prokash K. Chowrashi

Myofibrillogenesis in skeletal muscle cells

How are myofibrils assembled in skeletal muscles? The current authors present evidence that myofibrils assemble through a three-step model: premyofibrils to nascent myofibrils to mature myofibrils. This three-step sequence was based initially on studies of living and fixed cultured cells from cardiac muscle. Data from avian primary muscle cells and from a transgenic skeletal mouse cell line indicate that a premyofibril model for myofibrillogenesis also holds for skeletal muscle cells. Premyofibrils are characterized by minisarcomeres bounded by Z-bodies composed of the muscle isoform of alpha-actinin. Actin filaments are connected to these Z-bodies and to the mini-A-bands composed of nonmuscle myosin II filaments. Nascent myofibrils are formed when premyofibrils align and are modified by the addition of titin and muscle myosin II filaments. Mature myofibrils result when nonmuscle myosin II is eliminated from the myofibrils and the alpha-actinin rich Z-bodies fuse as the distance between them increases from 0.5 microm in premyofibrils to 2 to 2.5 microm in the mature myofibrils.

Assembly of Myofibrils in Cardiac Muscle Cells

How do myofibrils assemble in cardiac muscle cells? When does titin first assemble into myofibrils? What is the role of titin in the formation of myofibrils in cardiac muscle cells? This chapter reviews when titin is first detected in cultured cardiomyocytes that have been freshly isolated from embryonic avian hearts. Our results support a model for myofibrillogenesis that involves three stages of assembly: premyofibrils, nascent myofibrils and mature myofibrils. Titin and muscle thick filaments were first detected associated with the nascent myofibrils. The Z-band targeting site for titin is localized in the N-terminus of titin. This region of titin binds alpha-actinin and less avidly vinculin. Thus the N-terminus of titin via its binding to alpha-actinin, and vinculin could also help mediate the costameric attachment of the Z-bands of mature myofibrils to the nearest cell surfaces.

Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the hosts cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.

β‐thymosins from marine invertebrates: Primary structure and interaction with actin

The beta-thymosins are distributed throughout the vertebrate phyla, and all known vertebrate beta-thymosins bind actin monomers. To determine whether beta-thymosin-like peptides function as actin-binding proteins in invertebrates, we fractionated perchloric acid extracts of the gonads of both the sea urchin, Arbacia punctulata, and the scallop, Argopecten irradians, and screened the fractions for proteins which could be crosslinked to actin. In each case a peptide was isolated which crosslinks to actin from both rabbit skeletal muscle and scallop cross-striated adductor muscle; both peptides were sequenced and each was found to consist of 40 amino acid residues, compared with 41-43 residues for the vertebrate beta-thymosins. The sequences of the scallop and sea urchin beta-thymosins are 80% identical to each other, 75% identical to residues 1-40 of thymosin beta4, and 72-80% identical to residues 1-40 of other vertebrate beta-thymosins. The sea urchin peptide was found to inhibit actin polymerization and nucleotide exchange. The affinity of the sea urchin peptide for rabbit muscle actin is apparently lower than that of thymosin beta4, since about twice the concentration of sea urchin peptide is required to give inhibition of actin polymerization or nucleotide exchange equivalent to thymosin beta4.

Potential role of the EPEC translocated intimin receptor (Tir) in host apoptotic events

Apoptosis, or programmed cell death, is a well-ordered process that allows damaged or diseased cells to be removed from an organism without severe inflammatory reactions. Multiple factors, including microbial infection, can induce programmed death and trigger reactions in both host and microbial cellular pathways. Whereas an ultimate outcome is host cell death, these apoptotic triggering mechanisms may also facilitate microbial spread and prolong infection. To gain a better understanding of the complex events of host cell response to microbial infection, we investigated the molecular role of the microorganism Enteropathogenic Escherichia coli (EPEC) in programmed cell death. We report that wild type strain of EPEC, E2348/69, induced apoptosis in cultured PtK2 and Caco-2 cells, and in contrast, infections by the intracellularly localized Listeria monocytogenes did not. Fractionation and concentration of EPEC-secreted proteins demonstrated that soluble protein factors expressed by the bacteria were capable of inducing the apoptotic events in the absence of organism attachment, suggesting adherence is not required to induce host cell death. Among the known EPEC proteins secreted via the Type III secretion (TTS) system, we identified the translocated intimin receptor (Tir) in the apoptosis-inducing protein sample. In addition, host cell ectopic expression of an EPEC GFP-Tir showed mitochondrial localization of the protein and produced apoptotic effects in transfected cells. Taken together, these results suggest a potential EPEC Tirmediated role in the apoptotic signaling cascade of infected host cells.

LC2 involvement in the assembly of skeletal myosin filaments

The assembly of LC2-deficient myosin was studied under conditions where control and LC2-reassociated myosin assemble around the native length of about 1.5 microns. The aim of this work was to determine how loss of LC2 affects the assembly characteristics. The findings of this study can be summarized as follows: (a) LC2-deficient myosin assembles into two populations of filaments, one around 0.5 micron in length and the other around 1 micron in length. This suggests that loss of the LC2 perturbs the length-determining mechanism. (b) The population of filaments around 0.5 micron has a diameter around 14 nm and that around 1 micron a diameter around 22 nm. Neither diameter corresponds to the 18 nm obtained with the control and LC2-reassociated myosins, suggesting that the presence of LC2 may have a role in regulating the side-to-side assembly of the myosin rods. (c) Filaments assembled from LC2-deficient myosin tend to aggregate side-by-side, but not those assembled from control and LC2-reassociated myosin. (d) The presence of MgATP has no effect on the length distribution of LC2-deficient myosin filaments in contrast to the sharpening of the distribution observed with control and reassociated myosin.

The myosin filament. XII. Effect of MgATP on assembly

SummaryThe effect of MgATP on myosin filament assembly has been studied. Filaments were assembled by a standard dilution procedure involving two steps, dilution from 0.6 to 0.3m KCl and from 0.3 to 0.15m KC1 with a different rate of dilution in each step. This standard dilution procedure gives filaments which are structurally similar to native filaments in that they have a sharp length distribution around 1.5 μm, a diameter of 16 nm and they vary in length with KCl concentration in a similar manner to native filaments. The addition of 1mm MgATP leads to a sharpening of the length distribution around 1.5 μm without change in the 16 nm diameter. Filaments assembled by dialysis or by rapid dilution are not similarly affected by the presence of MgATP indicating that the standard dilution procedure produces filaments which are more closely similar to native filaments than those produced by these other methods. MgAMPPNP and magnesium pyrophosphate have the same effect as MgATP thus eliminating the possibility that phosphorylation of the myosin is involved in the effect. The effect of MgATP is not directly related to its binding to the active site of the myosin molecule since a 500∶1 mole ratio of MgATP to myosin is required for the effect. It is therefore likely that the effect of MgATP is related to other binding sites on the myosin molecule.The presence of MgATP leads to molecular rearrangements which finely tune the molecular organization of the filaments formed by the standard dilution procedurein vitro. It is likely that the MgATP present in living cells may similarly be responsible for the fine tuning of the molecular assembly of the myosin filaments to produce uniform lengthsin vivo.

Arg/Abl-binding protein, a Z-body and Z-band protein, binds sarcomeric, costameric, and signaling molecules.

ArgBP2 (Arg/Abl‐Binding Protein) is expressed at high levels in the heart and is localized in the Z‐bands of mature myofibrils. ArgBP2 is a member of a small family of proteins that also includes vinexin and CAP (c‐Cbl‐associated protein), all characterized by having one sorbin homology (SOHO) domain and three C‐terminal SH3 domains. Antibodies directed against ArgBP2 also react with the Z‐bodies of myofibril precursors: premyofibrils and nascent myofibrils. Expression in cardiomyocytes of plasmids encoding Yellow Fluorescent Protein (YFP) fused to either full length ArgBP2, the SOHO, mid‐ArgBP or the SH3 domains of ArgBP2 led to Z‐band targeting of the fusion proteins, whereas an N‐terminal fragment lacking these domains did not target to Z‐bands. Although ArgBP2 is not found in skeletal muscle cells, YFP‐ArgBP2 did target to Z‐bodies and Z‐bands in cultured myotubes. GST‐ArgBP2‐SH3 bound actin, α‐actinin and vinculin proteins in blot overlays, cosedimentation assays, and EM negative staining techniques. Over‐expression of ArgBP2 and ArgBP2‐SH3 domains, but not YFP alone, led to loss of myofibrils in cardiomyocytes. Fluorescence recovery after photobleaching was used to measure the rapid dynamics of both the full length and some truncated versions of ArgBP2. Our results indicate that ArgBP2 may play an important role in the assembly and maintenance of myofibrils in cardiomyocytes.

The Myosin Filament: XI Filament Assembly

The critical parameters required for the assembly of myosin filaments with a length distribution comparable to that for native myosin filaments were examined. It was found that: Two steps are required in the dilution of a myosin solution from 0.6M KCl to 0.15M KCl. In Step I the KCl concentration is reduced from 0.6 to 0.3M KCl and in Step II from 0.3 to 0.15M KCl. The rate of change of KCl required for Step I is different than that required for Step II. Increasing the total time of dilution in either Step I or II alone leads to an increase in length and a broadening of the length distribution. In Step I assembly of myosin molecules into nonsedimentable units occurs. These may be the basic units from which the filaments are assembled in Step II. Rapid dilution in Step I alone has no effect on the length distribution obtained at 0.15M KCl, but rapid dilution in Step II alone leads to short filaments (about 0.6 micron). Increasing the time of dilution in Step II alone to 3 hrs or 6 hrs gives a bimodal distribution in lengths with one peak at about 0.8 micron and the other at about 2.2 microns. The length distribution obtained at 0.15M KCl is not critically dependent on information contained in the portion of the filament previously assembled in Step II, but is critically dependent on the rate of change of KCl concentration during the assembly of the rest of the filament.

The myosin filament XIII. The sensitivity of LMM assembly to Mg·ATP

An LMM fragment (Mr 62,000) of myosin has been prepared which has aggregation properties that are sensitive to the presence of Mg.ATP. Aggregation of the LMM by reducing the ionic strength in the presence of 1 mM Mg.ATP produces non-periodic aggregates which gradually rearrange to paracrystals with a 43 nm axial repeat pattern. This fragment includes the C-terminal end of the myosin rod starting at residue 1376. Therefore, at least one of the Mg.ATP binding sites responsible for this effect is located somewhere along this region of the myosin rod. Although assembly of the rod fragment of myosin into paracrystals does not show sensitivity to Mg.ATP, assembly of intact myosin molecules to form filaments does show sensitivity to Mg.ATP. For myosin filaments, assembly initially gives a broad distribution around a mean length of 1.5 microns, which sharpens around the mean length with time. The rearrangement of the LMM rods and intact myosin molecules both induced by the presence of Mg.ATP are probably related. These findings highlight the complexity of the cooperative interactions between different portions of the myosin molecule that are involved in determining the assembly properties of the intact molecule.