J C Bulinski

Distinct populations of microtubules: Tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo

A unique post-translational modification of tubulin has previously been described in which a tyrosine residue is reversibly added to the C terminus of the alpha-tubulin subunit. We have prepared peptide antibodies that specifically react (shown by competitive immunoassay and Western blots) with the tyrosinated (Tyr) and nontyrosinated (Glu) forms of alpha-tubulin. Immunofluorescence with these antibodies demonstrated that the distributions of Tyr and Glu tubulin in fixed cells were markedly different. Tyr tubulin was found throughout the interphase network of microtubules and in the metaphase spindle, whereas Glu tubulin was present in a limited subset of interphase microtubules and was absent from the astral fibers of the metaphase spindle. Double immunofluorescence showed that Glu and Tyr microtubules comprised distinct subsets of the total cellular microtubules. These results suggest that tyrosination is involved in the establishment of separate populations of microtubules that may functionally distinct.

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the α- and γ-isoforms of actin☆

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.

Preparation and functional assay of pure populations of tyrosinated and detyrosinated tubulin.

Publisher Summary In all higher eukaryotes, a cycle of posttranslational modification yields two coexisting forms of α-tubulin, which differ by the presence or absence of a tyrosine residue at their C termini. The tyrosinating enzyme, tubulin tyrosine ligase (TTL), and to a lesser extent, the detyrosinating enzyme, tubulin carboxypeptidase (TCP), have been characterized. Dynamic microtubules (MTs) are enriched in tyrosinated (Tyr) α-tubulin subunits, while less dynamic (stable) MT subsets are enriched in detyrosinated (Glu) tubulin subunits. Although MTs enriched in Tyr or in Glu tubulin may be functionally distinct in vivo , the effect of these posttranslational modifications on tubulin function is currently unknown. An obvious approach to this dilemma is to study homogeneous preparations of Tyr and Glu tubulin in vitro. Preparation of Glu tubulin is easily achieved by digestion of tubulin or MT protein with pancreatic carboxypeptidase A (CPA). This chapter presents the procedures of taxol-dependent purification of Tyr and Glu tubulin from hela cells.

Peptide antibodies: new tools for cell biology.

The process of preparing antibodies against small peptide subsets of larger proteins is now a very routine and effective tool for cell biological investigations. Now that the identification of genes is commonplace, it is imperative to be able to identify, purify, and characterize the products of these genes. Antibodies against synthetic peptides will aid in discovering the elusive functions of these proteins. Over the past 5 years, peptide antibodies have contributed, and they will doubtless continue to contribute, to the identification of functional domains of proteins. Peptide antibodies provide a means for identifying functional domains conserved during the evolution of families of proteins, and for inhibiting specific functions of multifunctional proteins. Domain-specific antibodies have already increased the molecular resolution with which cell biologists can immunologically examine the function of cellular proteins. Finally, many proteins are now known to exist in subtly different forms, either as the products of separate genes or as the result of posttranslational modifications. Peptide antibodies allow molecular cell biologists, for the first time, to design antibodies for the specific assay of altered forms of a protein. Because they are amenable to specific immunolocalization of highly similar species, peptide antibodies can be considered to be subcellular probes of gene expression and posttranslational modification.

Changes in the Expression of β and Actins During Differentiation of PC 12 Cells

Abstract: Cells of the rat pheochromocytoma line PC 12 cease proliferation and develop neurites in response to nerve growth factor (NGF). Quantification of β and iso‐forms of nonmuscle actin in extracts of these differentiating cells showed that the β: ratio decreased from 1.30 ± 0.05 to 0.99 ± 0.05 after 6 days of NGF treatment. Cells treated with. N6, O2‐dibutyryl cyclic AMP (dbcAMP) also showed a shift in the ratio of β: isoforms, although few of these cells extended neurites. Administration of dbcAMP or both NGF and dbcAMP to cells accelerated the decrease in the β: actin isoform ratio relative to treatment with NGF alone. Those cells treated with both NGF and dbcAMP also showed an accelerated rate of neurite outgrowth. Suspension‐grown PC 12 cells treated with NGF showed neither an isoform ratio decrease nor neurite development. Our results suggest that either cyclic AMP may be a “second messenger” for NGF or it may effect the isoform ratio change by an independent mechanism. In addition, our data demonstrate an alteration in actin isoform expression, which accompanies the morphological differentiation of PC 12 cells.

Comparison of methods for tubulin quantitation in HeLa cell and brain tissue extracts

Abstract Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.