Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.

Abstract

A biochemical assay employing DNase-I affinity chromatography, two-dimensional peptide analysis, and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises^-1% of the total protein in homogenates of spermatocytes and spermatids . SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin . Under conditions that preserve native supramolecular states of actin, -80% of the spermatocyte actin is in a sedimentable form whereas only -30% of the spermatic! actin is sedimentable . These differences could be meaningful with regard to structural changes that occur during spermiogenesis . A comparative analysis of two-dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins . The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin .Actin has been found in a variety of nonmuscle cells (31) and, on that basis, many investigators have suggested that it may be a ubiquitous structural protein involved in several processes including cell locomotion (7,39), the maintenance of cell shape (6,35), and cell cleavage (33) . Included in the list of actincontaining cells are crane-fly gonial cells (spermatocytes, spermatids, and sperm of Nephrotoma suturalis) which have been reported by Forer and Behnke (11,12) to contain decorated microfilaments after treatment with glycerol and heavy meromyosin (19,20) . Those results raised the possibility that actin may be involved in the morphogenetic events of spermiogenesis . In addition, Forer and Behnke observed microfilaments in spindles of dividing spermatocytes (11) . That finding has been cited frequently as evidence in support of the idea that actin may play a role in the mechanism of chromosome transport (l0) .In an attempt to corroborate and extend the findings of Forer and Behnke (11, l2), we have conducted a biochemical analysis of the amount and supramolecular organization of actin in crane-fly gonial cells. Homogenates first were prepared in buffers that either stabilized actin pools in their native state or converted all cell actin to the monomeric DNase-binding THE JOURNAL OF CELL BIOLOGY " VOLUME 86 JULY 1980 315-325 © The Rockefeller University Press " 0021-9525/80/07/0315/11 $1 .00 form (3) and then were analyzed by a combination of DNase affinity chromatography and SDS polyacrylamide gel electrophoresis (PAGE) . This approach takes advantage of the specific interaction between actin and DNase I (27) and is especially applicable to cell types that contain limited amounts of actin . 43,000-dalton polypeptides that were isolated with DNase were characterized further by two-dimensional peptide mapping techniques .We have confirmed...

Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors

“…Fractions were collected, dialyzed, Iyophilized, and analyzed by NaDodSO4 electrophoresis.. Fig. 3 shows GAG, the 58,000-dalton polypeptide, and a portion ofthe actin eluting within the column near the retention volume of the column (fractions [10][11][12][13]. Thus, the three components behave as a complex on-both sucrose density gradient centrifugation and gel filtration.…”

“…S buffer was designated to stabilize actin microfilaments and to retard C-and F-actin interconversions (13). Triton/Pi/NaCl is a more commonly used extractant for making cytoskeletal preparations.…”

Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells.

“…for these studies were recovered from the peritoneal cavity after 7 d, washed with ice-cold Dulbecco's phosphate-buffered saline (DPBS), and resuspended in DPBS. Metabolic labeling with ['"C]glucosamine (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), ['"C]leucine (20,uCi), ['SS]methionine (25tuCi), or ["P]phosphate(25pCi) wasaccomplished by injection of the label in 0.1 ml of 0.9% NaCI into the peritoneal cavity of tumor-bearing rats^-16-18 h before sacrifice and recovery of the cells.…”

Malorientation and abnormal segregation of chromosomes during recovery from Colcemid and Nocodazole

“…inactivate Colcemid using the same procedure applied to Larvae for this study were selected from a laboratory colony of crane flies (Nephrotoma suturalis) using the critera of Strauch, Luna, and LaFountain [1980], and Forer [ 19821. Colcemid (Chemical Sales Division, CIBA Pharmaceutical, Summit, N.J.) was prepared in a stock solution at 2.7 X M (1 mg/ml) in 15 mM phospate buffer containing 5.3% (w/v) 95% ethanol and 10% (w/ v) propylene glycol. Nocodazole stock (Janssen Pharmaceutica, Life Sciences Products Division, Piscataway, N.J.) was dissolved in fresh dimethyl sulfoxide at a concentration of 10 mg/ml (3.3 X M).…”