Sucrose-induced spindle elongation in mitotic PtK-1 cells

Brief treatment of mitotic metaphase and anaphase PtK-1 cells with tissue culture medium containing 0.5 M sucrose resulted in spindle elongation without chromosome motion. Spindle birefringence also changed from a uniform appearance to one of highly birefringent bundles. Electron microscopic analysis indicated these birefringent bundles were composed of tightly packed arrays of spindle microtubules. No kinetochores could be seen following a 10 min sucrose treatment. Upon removal of sucrose, metaphase spindles returned to pretreatment lengths and the normal birefringence pattern returned. Reduction in spindle length could be temporally coupled with the reappearance of kinetochores and the reassociation of microtubules with these structures. In contrast to treated and released metaphase cells, anaphase spindles did not return to pretreatment lengths. Replacement of sucrose with medium showed the resumption of chromosome-to-pole motion within 2 min of sucrose removal. Chromosome motion could be correlated with the reappearance of kinetochores and kinetochore microtubules. These data have led us to postulate the existence of two microtubule continuums in the spindle and to discuss their roles in spindle organization and chromosome motion.     

Fluorescent analog of OSW-1 and its cellular localization

OSW-1 is a steroidal saponin, which has emerged as an attractive anticancer agent with highly cancer cell selective activity. A fluorescent analog was prepared from the natural product to analyze its cellular uptake and localization. We found that the fluorescent analog is rapidly internalized into cells and is primarily distributed in endoplasmic reticulum and Golgi apparatus.     

Cellular compartments in mitotic cells: ultrahistochemical identification of Golgi elements in PtK-1 cells

This investigation focuses on the identification, distribution, and transport of intracellular membrane systems during mitosis. The membranes of the Golgi apparatus can be identified cytochemically by staining for acid phosphatase (acPase) and thiamine pyrophosphatase (TPPase) activity. Using this approach we are able to study the disintegration of the Golgi apparatus during mitosis and to follow the dislocation as well as the organized reappearance of Golgi elements after the completion of mitosis. We are able to demonstrate that during mitosis the activity of both enzymes is strong enough to react with the substrate applied during the staining procedure. Furthermore, we observe a characteristic pattern of membrane distribution in mitotic cells. During interphase the TPPase reaction is characteristically limited to one or two cisternae of a dictyosomal stack. The acPase reaction stains the membranes of the total stack, of the GERL, of some vesicles and cisternae near the dictyosomes and lysosomes. After the mitotic breakdown of the dictyosomal stacks the forming vesicles still stain positively and are distributed over the entire cytoplasm. At late anaphase and early telophase the enzyme activity occurs not only in the reconstituting dictyosomes but also in the nuclear envelope and in some ER cisternae. The extended spectrum of membrane structures indicating Golgi enzyme activity becomes obvious. This phenomenon favors the idea that at least some functions of the Golgi apparatus persist during mitosis.     

Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells

Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.     

New aspects of sphingosine 1-phosphate signaling in mammalian cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid phosphate that binds to cell surface G-protein-coupled receptors (GPCR), but also can elicit intracellular actions. The role of S1P in cancer has been an area of significant interest and we have focused our research on two aspects that are of importance with respect to cancer. First, we have investigated how cross talk between S1P and growth factors might affect the pathophysiology of cancer cells. In this regard, we have demonstrated that S1P receptors function to re-programme the spatial signaling specificity of receptor tyrosine kinases and vice versa to modulate cell responses. Second, we have investigated spatial/temporal aspects of intracellular S1P signaling and how this might be de-regulated in cancer. This has involved studies on: (i) the interaction of sphingosine kinase 1 (which catalyses the phosphorylation of sphingosine to produce S1P) and phospholipase D in the Golgi apparatus linked to regulation of cell survival and (ii) the novel regulatory interaction between sphingosine kinase 1 and 2 and centrosomal S1P₅ receptor linked to the regulation of mitosis in mammalian cells including MDA-MB-231 breast cancer cells. Therefore, we have focused on novel aspects of spatial and temporal S1P signaling that might enable this bioactive lipid phosphate to exhibit normal and aberrant function in health and disease respectively.     

Fluorescent localization of the beta-adrenergic receptor on DDT-1 cells. Down-regulation by adrenergic agonists.

Continuous incubation of cultured cells with beta-adrenergic agonists results in the desensitization of adrenergic responsiveness accompanied by the down-regulation of cell surface beta-adrenergic receptors (beta AR). Previous studies have relied on measurements of ligand binding activity for the detection of the beta AR in the cell. In the present study, we have raised a monoclonal antibody to a synthetic peptide corresponding to amino acid numbers 226-239 of the hamster beta 2AR. This antibody was used to localize the beta AR in hamster smooth-muscle DDT-1 cells by immunofluorescence, without regard for the ability of the receptor to bind ligands. The beta AR was found to be localized primarily at the plasma membrane of these cells, with a nonhomogeneous pattern of distribution. A rapid loss of beta AR-specific immunofluorescence, which paralleled receptor down-regulation as measured by ligand-binding activity, was seen with beta-adrenergic agonists, but not with antagonists. In addition, a transient increase in fluorescence was observed after short times of exposure of the cells to agonists. This fluorescence increase may reflect a ligand-induced conformational change in the receptor.     

Analysis of centrosome localization of BRCA1 and its activity in suppressing centrosomal aster formation

BRCA1, a product of a familial breast and ovarian cancer susceptibility gene, localizes to centrosomes and physically interacts with γ-tubulin, a key centrosomal protein for microtubule nucleation and anchoring at centrosomes. Here, we performed a rigorous analysis of centrosome localization of BRCA1, and found that BRCA1 is specifically associated with mother centrioles in unduplicated centrosomes, and daughter centrioles acquire BRCA1 prior to initiation of duplication, and thus duplicated centrosomes are both bound by BRCA1. We further found that BRCA1 suppresses centrosomal aster formation. In addition, we identified a new domain of BRCA1 critical for γ-tubulin binding, which confers not only its localization to centrosomes, but also its activity to suppress centrosomal aster formation.     

Cytochemical localization of peroxidase activity in root cells

Summary The distribution of peroxidase in the apical 3 mm of pea roots has been investigated using the histochemical method employing 3,3-diaminobenzidine as a substrate. At the tissue level the enzyme is localized predominately in the root cap, epidermis, inner cortical cells, endodermis, phloem and maturing xylem. At the subcellular level peroxidase is found mainly in the intercellular regions of the cortex cell walls and in the cytoplasm and vacuoles of the steler cells. Root microbodies, unlike those of leaves, do not appear to be able to oxidize this substrate. The significance of these observations is discussed in relation to the validity of the technique and the proposed roles of the enzyme in cellular metabolism.     

Methodological aspects of the histochemical localization and activity of some cerebellar dehydrogenases

Summary In a detailed study focused on the methodological problems in dehydrogenase histochemistry [e.g., fixation, diffusion of enzymes and of reduced inermediates, conversion of NADPH and NADP to NADH and NAD, respectively, penetration of tetrazolium salt and formazan substantivity, nothing dehydrogenase reaction, use of exogenous CoQ₁₀ and of flavoprotein substitute (PMS)], the distribution and activity of succinate dehydrogenase, NAD(P)H-tetrazolium reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase (H and M types), and of l-glutamate dehydrogenase (E.C. 1.4.1.2 and E.C. 1.4.1.3) have been investigated in the rat cerebellum.It was evident from the study that reliable results could only be obtained if all the aforementioned factors had been considered. The image of actual concentration of SDH in the neuropil of the molecular layer could only be recorded by adding CoQ₁₀, while other structures exhibited greater balance between SDH and endogenous mitochondrial CoQ. Contrary to previous studies, a reversed localization of the activity of G-6-PDH and LDH was noticed. The elements of molecular and Purkinje layers were rich in G-6-PDH, while the granular layer was nearly depleted. The actual level of LDH could only be recorded if NADH-tetrazolium reductase was bypassed with PMS. The H and M types of LDH coexisted in the three cortical layers, the H type being prevalent and the M type attaining its highest level in synaptic glomeruli followed by the structures of the molecular layer and the Purkinje cells. High activity of GDH was noticed in Bergmann glia followed by synaptic glomeruli, while most other structures showed weak to moderate activity. The two GDH types coexisted in all structures showing activity, except for Bergmann cells, which only showed presence of the E.C. 1.4.1.3 type.Furthermore, Bergmann glia was exceptional by showing no activity of SDH and LDH, but strong activity of G-6-PDH and NADPH-tetrazolium reductase. The granular cells were exceptional by showing weak or no activity of all enzymes in question.     

Visual evoked potentials in the white New Zealand rabbit: source localization and normative aspects.

Normative data of visual evoked potentials were obtained from occipital screw electrodes referenced to a nasal electrode in 10 white New Zealand rabbits. Two time-bases were used (200 and 400 ms) in order to reliably identify both early components (of retinal and cortical origin), as well as late components. The putative origin of the single components was established by simultaneous recording of ERG and by stereotaxic recordings from the lateral geniculate body. Test-retest variability was measured repeating the recordings after 24 hours.     

New role for hPar-1 kinases EMK and C-TAK1 in regulating localization and activity of class IIa histone deacetylases

Class IIa histone deacetylases (HDACs) are found both in the cytoplasm and in the nucleus where they repress genes involved in several major developmental programs. In response to specific signals, the repressive activity of class IIa HDACs is neutralized through their phosphorylation on multiple N-terminal serine residues and 14-3-3-mediated nuclear exclusion. Here, we demonstrate that class IIa HDACs are subjected to signal-independent nuclear export that relies on their constitutive phosphorylation. We identify EMK and C-TAK1, two members of the microtubule affinity-regulating kinase (MARK)/Par-1 family, as regulators of this process. We further show that EMK and C-TAK1 phosphorylate class IIa HDACs on one of their multiple 14-3-3 binding sites and alter their subcellular localization and repressive function. Using HDAC7 as a paradigm, we extend these findings by demonstrating that signal-independent phosphorylation of the most N-terminal serine residue by the MARK/Par-1 kinases, i.e., Ser155, is a prerequisite for the phosphorylation of the nearby 14-3-3 site, Ser181. We propose that this multisite hierarchical phosphorylation by a variety of kinases allows for sophisticated regulation of class IIa HDACs function.     

Arachidonate 5-lipoxygenase of porcine pancreas: its localization in acinar cells

Arachidonate 5-lipoxygenase has been found so far in various types of leukocyte. When a homogenate of porcine pancreas was incubated with arachidonic acid, 5-hydroxy-6,8,11,14-eicosatetraenoic acid was predominantly produced concomitant with small amounts of compounds derived from leukotriene A4. After differential centrifugation of the homogenate, the 5-lipoxygenase activity was found predominantly in the 1000 x g pellet and 105,000 x g supernatant. When porcine pancreas was investigated immunohistochemically with anti-5-lipoxygenase antibody, Langerhans islets were unstained, and infiltration of 5-lipoxygenase-positive leukocytes was hardly observed. In contrast, acinar cells were positively stained. Immunoelectron microscopy demonstrated the localization of the enzyme along the nuclear membranes of the acinar cells.