Marvin H. Stromer

Identification of the 30 kDa polypeptide in post mortem skeletal muscle as a degradation product of troponin-T.

Although a 30 kDa polypeptide frequently is seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of post mortem (pm) skeletal muscle and in turn is used as an indicator of proteolysis, its origin has not been conclusively identified. We used antibodies to selected myofibrillar proteins, including some known to be degraded pm, to identify this polypeptide. The left side of eight beef carcasses was electrically stimulated (ES) within 1 h after slaughter, and the right side served as the non-stimulated (NS) control. The longissimus lumborum (LL) muscle was removed from the carcass at 24 h pm and was stored at 2 degrees C. Myofibrils were prepared from the LL muscle immediately after stimulation (0 day) and from the stored muscle sample at 1, 3, 7, 14 and 28 days pm for analysis of SDS-PAGE and Western blots. By SDS-PAGE, troponin-T (TN-T) decreased in amount more rapidly pm in ES samples than in NS samples. By SDS-PAGE, a 30 kDa band increased and became a prominent band by 7 days pm in both NS and ES samples. A monoclonal antibody (mAb) to TN-T labeled purified TN-T, as well as the TN-T in myofibrils, a prominent 30 kDa polypeptide and a family of lower molecular mass polypeptides in pm muscle. This mAb also labeled a 30 kDa band that had been electrophoretically purified from pm muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles

The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remains largely unknown. Zymogen granules (ZGs), the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a Gαi3 protein. Evidence is presented here that the water channel aquaporin-1 (AQP1) is present in the ZG membrane and participates in rapid GTP-induced vesicular water gating and swelling. Isolated ZGs exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of ZGs with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulatable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxyl-terminal domain of AQP1 blocks GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the ZG membrane is involved in basal as well as GTP-induced rapid gating of water in ZGs of the exocrine pancreas.

Structure and Composition of the Fusion Pore

Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, alpha-fodrin and the calcium channels alpha1c and beta3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.

STRUCTURE AND DYNAMICS OF THE FUSION PORE IN LIVE CELLS

Atomic force microscopy reveal pit‐like structures typically containing three or four, ∼150nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion. It is hypothesized that depressions are the fusion pores, where membrane‐bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane‐bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase‐specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High‐resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.

Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture.

A model of the structure of vertebrate Z-lines and Z-line analogs is introduced and supported by evidence from electron microscope studies of wide Z-lines (rat and feline soleus, and feline and canine cardiac muscles), narrow Z-lines (guppy, newt and frog skeletal muscles), and Z-rods (from a patient with nemaline myopathy and from cardiac muscles of aged dog). The model is based on a pair of Z-filaments (termed a Z-unit), which are linked near their centers at a 90 degrees angle and form bridges between neighboring antipolar thin (actin) filaments. A square lattice of four Z-filament pairs (the basic structure of the Z-line, termed a Z-line unit) defines the geometrical position of the I-square unit. In this native state of the Z-line, small square and large square net forms appear in cross-section. Other cross-sectional patterns of Z-lines, including basket-weave and diagonal-square net patterns, can be explained by detachment of the Z-filament from the Z-filament binding region within each Z-filament pair due to chemical or physical stress. Dissection of Z-lines and Z-line analogs with calcium-activated neutral protease provides evidence that the width of all wide Z-line structures is determined by the amount of overlap of antipolar thin filaments from adjacent sarcomeres. Longitudinal patterns of narrow and wide Z-lines are shown and described in relation to the model. To test the proposed model, the dynamics of the Z-line unit structure were computer-simulated. An attempt was made to correlate longitudinal (z direction) and cross-sectional (x and y directions) patterns and to determine the amount of movement of thin or Z-filaments that is required to explain the diversity observed in cross-sectional patterns of Z-lines. The computer simulations demonstrated that the structural transitions among the small square, and therefore large square net, as well as basket-weave and diagonal-square net forms seen in cross-sections could be caused by movements of thin filaments less than 10 nm in any direction (x, y or z).(ABSTRACT TRUNCATED AT 400 WORDS)

Reconstituted Fusion Pore

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.

Studies on purified α-actinin: II. Electron microscopic studies on the competitive binding of α-actinin and tropomyosin to Z-line extracted myofibrils☆

Abstract α-Actinin forms an extensive network of cross-connections between thin filaments in the I-band when incubated with Z-line-extracted fibrils at 0 °C; this result may be due to the very high affinity of α-actinin for actin at 0 °C rather than indicating any other in situ localization of α-actinin in addition to the Z-line. By incubating low-ionic-strength extracted, glycerinated fiber bundles in highly purified α-actinin at 37 °C, cross-connections are predominately limited to the Z-line terminus of thin filaments. Incubation of extracted fiber bundles in tropomyosin at 0 °C results in no visible binding, but at 37 °C circular tufts are bound in three I-band zones. Incubating Z-line-extracted fibers in equal quantities of α-actinin and tropomyosin at 0 °C shows that tropomyosin does not interfere with α-actinin binding. At 37 °C, tropomyosin reduces the number of α-actinin cross-connections, but still binds in the three characteristic I-band zones. Unextracted fibers incubated in α-actinin at 0 °C also show the heavy network of I-band cross-connections; this indicates that at 0 °C, α-actinin can displace tropomyosin from the thin filament in vitro.

Purification and properties of α-actinin from rabbit skeletal muscle☆

Abstract The 6-S α-actinin species can be purified from a P15–25 α actinin fraction by DEAE-cellulose chromatography. The resulting P15–25 (DEAE) fraction is eluted as a single peak upon rechromatography on 4% agarose or DEAE-cellulose columns, although the rechromatography on DEAE-cellulose removes a very small amount of aggregates from the P15–25 (DEAE) fraction. Sedimentation diagrams of the P15–25 (DEAE) fraction show that approx. 85% of the protein in this fraction sediments with an s°20,w = 6.23 and about 10–15% sediments with an observed s value of 9.1. The 9.1-S component may be an aggregate of the 6.2-S species. The P15–25 (DEAE) fraction exhibits 2–3-fold higher specific activity in the turbidity assay of α-actinin activity than the original P15–25 fraction. Amino acid composition of the P15–25 (DEAE) fraction is clearly different from the amino acid composition of actin, demonstrating that α-actinin is a separate protein component of the myofibril and is not simply an unusual form of denatured actin. These results also show that the 6-Sα actinin species does not exhibit marked aggregating tendencies and that the large aggregates prevalent in earlier α-actinin preparations were probably due to the presence of denatured actin in these preparations. By using purified α-actinin, it was shown that the stoichiometry of the α-actinin-F-actin interaction is 0.41 parts of P15–25 (DEAE) to 1 part of F-actin. This corresponds to a molecular ratio of one α-actinin to ten G-actin monomers. Tropomyosin and α-actinin compete for the same or closely located binding sites on actin, but at 0°, α-actinin appears able to displace tropomyosin from F-actin. The presence of α-actinin was demonstrated in low-ionic-strength extracts of glycerinated fiber bundles of rabbit psoas muscle; this extraction causes removal of both Z-lines and M-lines. Incubation of Z-line-extracted fibrils with the P15–25 α-actinin fraction caused moderate reconstitution of Z-lines in these fibrils. Since the Z-line constitutes about 6% of the dry mass of the myofibril, but α-actinin makes up only about 1% of the myofibrillar protein, the Z-line is probably composed of substances in addition to α-actinin.

Nemaline myopathy rod bodies

Ca2+-activated protease (CAF) digestion of glycerinated nemaline myopathy muscle removed the electron-dense material covering rods and Z-lines and exposed longitudinal backbone filaments, 6-7 nm wide, which span the lengths of the original rods. Decoration of the exposed filaments (which are responsible for the periodicity parallel to the long axis of intact nemaline rods) with heavy meromyosin (HMM) proved they are actin filaments. After CAF treatment, cross-striated periodical patterns in longitudinal sections and Z-filament-like proteins connecting actin filaments seen in cross-section disappeared. This suggests that alpha-actinin may be involved in formation of this pattern because of the specificity of CAF toward alpha-actinin. Gel electrophoresis of CAF-treated nemaline muscle showed that most alpha-actinin is released into the supernatant, whereas the residue is mainly actin and myosin. Electron microscope examination of longitudinal sections of intact rods shows an oblique filament pattern, thin (7 nm) lines, thick (11 nm) lines, and an amorphous-appearance previously observed in normal Z-lines, patterns observed depend on sectioning angle and section thickness. In cross-section, rods show small square net (SS) and basket-weave (BW) forms. The SS form predominates and coexistence of the 2 forms, which also occur in normal Z-lines, is observed. Results support the idea that rods are lateral polymers of Z-line units. We think that the length of rods, as well as the width of Z-lines, is determined by the amount of overlap of actin filaments of opposite polarity. Initiation of rod formation may be due to deregulation of actin filament length.

Purified desmin from adult mammalian skeletal muscle: A peptide mapping comparison with desmins from adult mammalian and avian smooth muscle

Abstract Comparative one-dimensional peptide maps were prepared by the electrophoresis of digests derived from treatment of desmins with Ca 2+ -activated muscle protease, trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. Desmins from adult mammalian skeletal and smooth muscles were very similar. Avian smooth muscle desmin, although homologous with respect to many peptides, was different from the mammalian smooth and skeletal desmins. The amino acid compositions of the three desmins were quite similar.