Subcellular distribution of calcium-binding proteins and a calcium- ATPase in canine pancreas [published erratum appears in J Cell Biol 1990 Oct;111(4):1726]

Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with "Stains-All." Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER.     

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.     

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 x 10(-7) cm2/S, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 x 10(-7) cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 x 10(-8) cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITC-dextran indicated that FITC-dextran was relatively excluded excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.     

Mammalian nonsarcomeric myosin regulatory light chains are encoded by two differentially regulated and linked genes [published erratum appears in J Cell Biol 1990 Nov;111(5 Pt 1):2207]

The myosin 20,000-D regulatory light chain (RLC) has a central role in smooth muscle contraction. Previous work has suggested either the presence of two RLC isoforms, one specific for nonmuscle and one specific for smooth muscle, or the absence of a true smooth muscle-specific isoform, in which instance smooth muscle cells would use nonmuscle isoforms. To address this issue directly, we have isolated rat RLC cDNAs and corresponding genomic sequences of two smooth muscle RLC based on homology to the amino acid sequence of the chicken gizzard RLC. These cDNAs are highly homologous in their amino acid coding regions and contain unique 3'-untranslated regions. RNA analyses of rat tissue using these unique 3'-untranslated regions revealed that their expression is differentially regulated. However, one cDNA (RLC-B), predominantly a nonmuscle isoform, based on abundant expression in nonmuscle tissues including brain, spleen, and lung, is easily detected in smooth muscle tissues. The other cDNA (RLC-A; see Taubman, M., J. W. Grant, and B. Nadal-Ginard. 1987. J. Cell Biol. 104:1505-1513) was detected in a variety of nonmuscle, smooth muscle, and sarcomeric tissues. RNA analyses comparing expression of both RLC genes with the actin gene family and smooth muscle specific alpha-tropomyosin demonstrated that neither RLC gene was strictly smooth muscle specific. RNA analyses of cell lines demonstrated that both of the RLC genes are expressed in a variety of cell types. The complete genomic structure of RLC-A and close linkage to RLC-B is described.     

Midzone microtubule bundles are continuously required for cytokinesis in cultured epithelial cells [published erratum appears in J Cell Biol 1996 Dec;135(6 Pt 1):1679]

The current model of cytokinesis proposes that spindle poles and associated microtubules determine the cleavage plane, and, once the signal has been delivered to the cortex, the entire mitotic apparatus can be removed without affecting cell division. While supported by compelling data from Echinoderm embryos, recent observations suggest that the model may not be universally applicable. In this study, we have examined the relationship(s) among microtubules, chromosomes, and cleavage activity in living normal rat kidney (NRK) cells with multipolar mitotic figures. We found that cleavage activity correlated with the distribution of midzone microtubule bundles and Telophase Disc 60 protein (TD60) rather than the position of spindle poles. In addition, reduction of midzone microtubules near the cortex, by either nocodazole treatment or spontaneous reorganization in tripolar cells, caused inhibition or regression of furrowing. These results demonstrate that continuous interaction between midzone microtubule bundles and the cortex is required for successful cleavage in tissue culture cells.     

A 13-A map of the actin-scruin filament from the limulus acrosomal process [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1621]

We have determined the structure of the actin-scruin filament to 13-A resolution using a combination of low-dose EM and image analysis. The three-dimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2(+)- ADP actin filaments in the absence of Ca2+. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross- links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains III and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin.     

Regulation of fibronectin receptor distribution [published erratum appears in J Cell Biol 1992 Jul;118(2):491]

Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition of 12-o-tetradecanoylphorbol 13-acetate (TPA; 0.1 microM) induces irreversible differentiation into adherent, non-dividing, multinucleated cells. TPA-treated cells bear surface antigens typical of fetal osteoclasts, degrade 45Ca-labeled devitalized bone particles, display tartrate-resistant acid phosphatase in both mononuclear and multinuclear cells and receptors for calcitonin. Calcitonin increases intracellular cAMP accumulation in TPA-treated cells. TPA-treated cells show some ultrastructural features of osteoclasts as evidenced by transmission EM. These results indicate that FLG 29.1 cells may represent an osteoclast committed cell population, which upon induction with TPA acquire some morphological, phenotypical, and functional features of differentiated osteoclasts.     

Analysis of lateral redistribution of a plasma membrane glycoprotein- monoclonal antibody complex [corrected] [published erratum appears in J Cell Biol 1988 Apr;106(4):following 1403]

The lateral redistribution of a major murine glycoprotein, GP80, was studied on locomoting fibroblasts, using rhodamine-conjugated mAbs and ultralow light level digitized fluorescence microscopy. Confirming an earlier study (Jacobson, K., D. O'Dell, B. Holifield, T.L. Murphy, and J. T. August. 1984. J. Cell Biol. 99:1613-1623), the distribution of GP80 was coupled with cell locomotion; motile cells exhibited a gradated distribution of the GP80-mAb complex over the cell surface, increasing from the front to the rear, whereas stationary cells exhibited a nearly uniform GP80 distribution. By monitoring locomoting single cells, we found the gradated fluorescence distribution to be maintained as an approximate steady state. Newly extended leading edges were almost devoid of the fluorescence labeling. This was strikingly demonstrated in prechilled cells in which the extension of fluorescence-free leading edges caused a pronounced boundary between fluorescent and nonfluorescent zones. Subsequently this boundary eroded gradually in a manner consistent with diffusional relaxation. Evidence indicated that the GP80 redistribution was primarily caused by the lateral motion of GP80 in the plasma membrane and not via intracellular membrane traffic. Two cell locomotion models which, in principle, could account for the GP80 redistribution were tested: the retrograde lipid flow (RLF) model (Bretscher, M. S., 1984. Science (Wash. DC). 224:681-686) and an alternative hypothesis, the retraction-induced spreading (RIS) model. The predictions of these models were stimulated by computer and compared with experiment to assess which model was more appropriate. Whereas both models predicted steady-state gradients similar to the experimental result, only the RIS model predicted the lack of retrograde movement of the fluorescent boundary.     

Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae [published erratum appears in J Cell Biol 1995 Oct;131(2):561]

In many eucaryotic cells, the midzone of the mitotic spindle forms a distinct structure containing a specific set of proteins. We have isolated ASE1, a gene encoding a component of the Saccharomyces cerevisiae spindle midzone. Strains lacking both ASE1 and BIK1, which encodes an S. cerevisiae microtubule-associated protein, are inviable. The analysis of the phenotype of a bik1 ase1 conditional double mutant suggests that BIK1 and ASE1 are not required for the assembly of a bipolar spindle, but are essential for anaphase spindle elongation. The steady-state levels of Ase1p are regulated in a manner that is consistent with a function during anaphase: they are low in G1, accumulate to maximal levels after S phase and then drop as cells exit mitosis. Components of the spindle midzone may therefore be required in vivo for anaphase spindle movement. Additionally, anaphase spindle movement may depend on a dedicated set of genes whose expression is induced at G2/M.     

Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells [published erratum appears in J Cell Biol 1990 Mar;110(3):859]

Addition of EGF to human epidermoid carcinoma A431 cells increases the rate of fluid-phase pinocytosis 6-10-fold as measured by horseradish peroxidase uptake (Haigler, H.T., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:82-90). We show here that in the absence of extracellular Na+ or in the presence of amiloride the stimulation of pinocytosis by EGF is substantially reduced. Amiloride had no effect on the endocytosis of EGF itself or of transferrin, demonstrating that the receptor-mediated endocytotic pathway operated normally under conditions that blocked stimulated pinocytosis. Amiloride blocked EGF- stimulated pinocytosis in both HCO3(-)-containing and HCO3(-)-free media. The EGF-stimulated pinocytotic activity can frequently be localized to areas of the cell where membrane spreading and ruffling are taking place. These results demonstrate that (a) EGF induces a distinct amiloride-sensitive endocytotic pathway on A431 cells; (b) occupied EGF receptors do not utilize this pathway for their own entry; (c) endocytosis of occupied EGF receptors is not in itself sufficient to stimulate pinocytosis.     

Primary structure of the brain alpha-spectrin [published erratum appears in J Cell Biol 1989 Mar;108(3):following 1175]

We have determined the nucleotide sequence coding for the chicken brain alpha-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The alpha-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly alpha-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with alpha-actinin, (b) two typical EF-hand (i.e., Ca2+-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that alpha-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte alpha-spectrin is divergent from the other spectrins.     

Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p [published erratum appears in J Cell Biol 1996 Dec;135(6 Pt 2):1925]

The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small- budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal- Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.     

Expression of normal and mutant avian integrin subunits in rodent cells [published erratum appears in J Cell Biol 1989 Oct;109(4 Pt 1):1187]

We describe the expression of the beta 1 subunit of avian integrin in rodent cells with the purpose of examining the structure-function relationships of various domains within this subunit. The exogenous subunit is efficiently and stably expressed in 3T3 cells, and it forms hybrid heterodimers with endogenous murine alpha subunits, including alpha 3 and alpha 5. These heterodimers are exported to the cell surface and localize in focal contacts where both extracellular matrix and cytoskeleton associate with the plasma membrane. Hybrid heterodimers consisting of exogenous beta 1 and endogenous alpha subunits bind effectively and specifically to columns of cell-binding fragments of fibronectin. The exogenous avian beta 1 subunit appears to function as well as its endogenous murine equivalent, consistent with the high degree of conservation noted previously for integrins. In contrast, expression of a mutant form of avian integrin beta 1 subunit lacking the cytoplasmic domain produces hybrid heterodimers which, while efficiently exported to the cell surface and still capable of binding fibronectin, do not localize efficiently in focal contacts. This further implicates the cytoplasmic domain of the beta 1 subunit in interactions required for cytoskeletal organization.     

Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons [published erratum appears in J Cell Biol 1990 May;110(5):1845]

A novel gene of the calmodulin superfamily, encoding a 29-kD neuronal protein here named "calretinin," has been isolated as a cDNA clone from chick retina. The encoded sequence includes four putative calcium-binding sites and a fusion protein binds calcium. The most similar protein known is the 28-kD intestinal calcium-binding protein, calbindin (58% homology). Both genes date from before the divergence of chicks from mammals. The distribution of calretinin and calbindin mRNAs in chick tissues has been mapped using RNA gel blots and in situ hybridization. RNAs from both genes are abundant in the retina and in many areas of the brain, but calretinin RNA is absent from intestine and other nonneural tissues. Calretinin and calbindin are expressed in different sets of neurons throughout the brain. Calretinin RNA is particularly abundant in auditory neurons with precisely timed discharges.     

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation [published erratum appears in J Cell Biol 1998 Dec 28;143(7):following 2090]

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.     

Laminin forms an independent network in basement membranes [published erratum appears in J Cell Biol 1992 Jun;118(2):493]

Laminin self-assembles in vitro into a polymer by a reversible, entropy-driven and calcium-facilitated process dependent upon the participation of the short arm globular domains. We now find that this polymer is required for the structural integrity of the collagen-free basement membrane of cultured embryonal carcinoma cells (ECC) and for the supramolecular organization and anchorage of laminin in the collagen-rich basement membrane of the Engelbreth-Holm-Swarm tumor (EHS). First, low temperature and EDTA induced the dissolution of ECC basement membranes and released approximately 80% of total laminin from the EHS basement membrane. Second, laminin elastase fragments (E4 and E1') possessing the short arm globules of the B1, B2, and A chains selectively acted as competitive ligands that dissolved ECC basement membranes and displaced laminin from the EHS basement membrane into solution. The fraction of laminin released increased as a function of ligand concentration, approaching the level of the EDTA-reversible pool. The smaller (approximately 20%) residual pool of EHS laminin, in contrast, could only be effectively displaced by E1' and E4 if the collagenous network was first degraded with bacterial collagenase. The supramolecular architecture of freeze-etched and platinum/carbon replicated reconstituted laminin gel polymer, ECC, and collagenase-treated EHS basement membranes were compared and found to be similar, further supporting the biochemical data. We conclude that laminin forms a network independent of that of type IV collagen in basement membranes. Furthermore, in the EHS basement membrane four-fifths of laminin is anchored strictly through noncovalent bonds between laminin monomers while one-fifth is anchored through a combination of these bonds and laminin-collagen bridges.