Cell Type-specific Distribution Research Articles

Cell cycle-dependent transcriptional and proteolytic regulation of FtsZ in Caulobacter.

In the differentiating bacterium Caulobacter crescentus, the cell division initiation protein FtsZ is present in only one of the two cell types. Stalked cells initiate a new round of DNA replication immediately after cell division and contain FtsZ, whereas the progeny swarmer cells are unable to initiate DNA replication and do not contain FtsZ. We show that FtsZ expression is controlled by cell cycle-dependent transcription and proteolysis. Transcription of ftsZ is repressed in swarmer cells and is activated concurrently with the initiation of DNA replication. At the end of the DNA replication period, transcription of ftsZ decreases substantially. We show that the global cell cycle regulator CtrA is involved in the cell cycle control of ftsZ transcription. CtrA binds to a site that overlaps the ftsZ transcription start site. Removal of the CtrA-binding site results in transcription of the ftsZ promoter in swarmer cells. Decreasing the cellular concentration of CtrA increases ftsZ transcription and conversely, increasing the concentration of CtrA decreases ftsZ transcription. Because CtrA is present in swarmer cells, is degraded at the same time as ftsZ transcription begins, and reappears when ftsZ transcription decreases at the end of the cell cycle, we propose that CtrA is a repressor of ftsZ transcription. We show that proteolysis is an important determinant of cell type-specific distribution and cell cycle variation of FtsZ. FtsZ is stable when it is synthesized and assembles into the cytokinetic ring at the beginning of the cell cycle. After the initiation of cell division, the rate of FtsZ degradation increases as both the constriction site and the FtsZ ring decrease in diameter. When ftsZ is expressed constitutively from inducible promoters, the abundance of FtsZ still varies during the cell cycle. The coupling of transcription and proteolysis to cell division ensures that FtsZ is inherited only by the progeny cell that will begin DNA replication immediately after cell division.

Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing.

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin- (HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1-56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.

Spatial repositioning of centromeric domains during regrowth of axons in nuclei of murine dorsal root ganglion neurons in vitro.

Specific chromatin domains within interphase nuclei are organized in cell type specific distributions and are rearranged in association with changes in cell function. Axotomy leads to changes in gene expression. Dorsal root ganglion (DRG) neurons in vitro are a model for axotomy because they are detached from their axons in preparation for the culturing procedure. In a test of the hypothesis that neurons regrowing in vitro undergo rearrangement of specific chromatin domains, changes in the distribution of centromere-associated kinetochores proteins within DRG neurons were assessed as a function of time in vitro. Camparison of the kinetochore distributions in neurons in situ to those 24 h after placement into culture showed that the mean proportion (+/-S.E.M.) of kinetochore signals in the karyoplasm decreased from 41.0 +/- 1.8% to 28.6 +/- 3.3%, while the proportion at the nucleolus increased from 35.2 +/- 2.0% to 48.4 +/- 2.9%. This indicated redistribution of centromeric domains to the nucleolus. Between 1 day and 16 days in vitro, signals were redistributed to the nuclear periphery, indicated by an increase in the proportion of signals in this nuclear compartment from 23.0 +/- 4.3% to 37.6 +/- 3.4% and a decrease in the proportion of signals from 48.4 +/- 2.9% to 23.0 +/- 2.3% at the nucleolus. The results indicate that neurite regrowth following axotomy is associated with changes in nuclear topology. The reorganization that occurs within 24 h is speculated to be associated with a recapitulation of a cytoskeletal development program, while later changes in centromeric distributions may be related to cues elicited by in vitro conditions.

Localization of O6-alkylguanine transferase in cancer susceptible cells of human female breast.

The cell type specific distribution of O6-alkylguanine-DNA-alkyltransferase (AGT) protein was assessed using immunohistochemical localization in human female breast tissue sections and biochemical quantitation of fractionated cell extracts. The results demonstrated that the AGT protein is predominantly localized in luminal epithelial and myoepithelial cells of the intralobular mammary ducts. Western blot analysis revealed that the AGT level in epithelial cell rich organoid fraction was substantially higher than the whole tissue and fibro-collagenous stromal cell fraction of the normal breast. The quantitative activity measurements confirmed the occurrence of a statistically significant 2.7-fold (P = 0.05) and 4.0-fold (P = 0.04) enriched AGT activity in extracts prepared from the organoids compared to the whole tissue homogenate and fractionated stromal cells, respectively. The results suggest that the invariably high AGT level in malignant compared to the normal breast tissue could be due to AGT accumulation in luminal epithelial and myoepithelial cell mass, increasing as a consequence of the uncontrolled proliferation and differentiation of ductal cells in invasive carcinoma.

Integrins: role in cell adhesion and communication.

Adhesive interactions are crucial for the integrity and function of all cells and tissues. As one of the major families of cell adhesion receptors, the integrins have been the focus of scientific interest for more than a decade. The resulting studies have tremendously enhanced the understanding of integrin-mediated adhesive interactions and have unveiled novel integrin functions in the cytoskeletal organization of microfilaments and in the activation of diverse signaling pathways. These functions are critically involved in the regulation of multiple processes, such as tissue development, inflammation, tumor cell growth and metastasis, and programmed cell death. The global view of integrin receptor biology has radically changed and has become much more subtle and elaborate. The enormous complexity of integrin function is determined by the heterodimeric formation of more than 20 functional integrin receptors, the cell type-specific distribution, the receptor activation state, the presence of different activation and deactivation signals, and the subsequent employment of distinct cytoskeletal and signaling complexes within a more dimensional network of time and space. This article summarizes the structural and functional properties of the integrin receptors and emphasizes some of the major achievements made in the past to enhance the understanding of integrin biology.

Subunits of soluble guanylyl cyclase in rat and guinea-pig sensory ganglia

Soluble guanylyl cyclase is a heterodimeric (α, β) enzyme generating the second messenger, cGMP, upon activation by the gaseous messenger, nitric oxide. The occurrence and distribution of α 1 -, α 2 -, β 1 - and β 2-subunits were investigated in trigeminal and dorsal root ganglia on the mRNA and the protein level. Reverse transcription PCR analysis demonstrated mRNA coding for α 1 -, α 2 -, and β 1-subunits in guinea-pig trigeminal and dorsal root ganglia. In agreement with these data, immunoreactivity to the α 1-subunit was found in satellite and Schwann cells, while α 2-subunit immunoreactivity was localized to axons of large diameter. The distribution of the β 1-subunit could not be studied on the protein level since the antiserum was ineffective in immunohistochemistry. However, previous studies and the RT-PCR data argue in favour of α 1/β 1 - and α 2/β 1-heterodimerization and colocalization. In both species, β 2-subunit immunoreactivity was confined to neuronal perikarya, primarily of large diameter. Although these results were obtained with two different antibodies detected against different epitopes, the corresponding mRNA could not be detected by RT-PCR analysis. The reason for this discrepancy remains unclear, at present, but could be explained by a variant β 2 - or highly homologous as yet unidentified β-subunit. This study demonstrates the presence of soluble guanylyl cyclase in sensory ganglia with a differential, cell type-specific distribution of the individual subunits.

MAP2a, an alternatively spliced variant of microtubule-associated protein 2.

MAP2, a dendritically localized microtubule-associated protein (MAP), consists of a pair of high molecular mass (280 kDa) polypeptides, MAP2a and MAP2b, and several low molecular mass (70 kDa) proteins called MAP2c. Although MAP2b and MAP2c have been shown to arise via alternative splicing. It was not clear whether MAP2a is also created by alternative splicing or by posttranslational modification. Using epitope peptide mapping, we have demonstrated that an element specific to MAP2a is situated at its N-terminal end. A cDNA clone from an adult rat brain library was found to contain an additional 246 nucleotides situated at the 5' end of the 9-kb MAP2 mRNA. Antibodies generated against the encoded protein sequence recognize specifically MAP2a in rat brain homogenates. Moreover, although MAP2a, like MAP2b, is found in dendrites and cell bodies, its temporal appearance and cell type-specific distribution in rat brain differs from MAP2b.

Two Golgi integral membrane proteins (GIMPS) exhibit region- and cell type-specific distribution in the epididymis of the adult rat.

The epididymis participates in the post-testicular maturation and storage of spermatozoa by secreting proteins into the tubule lumen in a region-specific fashion. The underlying molecular mechanisms leading to biogenesis of these region-specific differences, however, are not known, although components of the Golgi complex membrane container must undoubtedly be intimately involved. Two monoclonal antibodies raised against Golgi integral membrane proteins, recognizing either the cis (GIMPc) or trans Golgi (GIMPt) cisternae, were used as molecular probes of these regions to begin the characterization of the Golgi complex of in vivo and in vitro epididymal cells. Immunolocalization of GIMPs was performed on frozen sections and in cultured cells using biotin-streptavidin-peroxidase immunocytochemistry. In tissue sections, immunostaining of GIMPt was extremely robust in the supranuclear cytoplasm throughout the epididymis. In contrast, no GIMPc immunostaining was detected in the initial segment or in clear cells of the distal caput, corpus, and cauda. Immunodetection of GIMPc and GIMPt in epididymal cells in vitro revealed a reticular, perinuclear pattern, and NH4Cl treatment preferentially disrupted the GIMPt immunolocalization. These results characterizing the molecular components of the Golgi complex will form the basis of additional studies to gain further insight into mechanisms leading to generation of regional differences in epididymal function.

Distribution of transforming growth factor alpha precursors in the mouse uterus during the periimplantation period and after steroid hormone treatments.

Temporal and cell-type specific distribution of transforming growth factor alpha (TGF alpha) precursor (proTGF alpha) was examined in the mouse uterus during the periimplantation period, and after steroid hormone treatments of ovariectomized adult mice by immunohistochemistry using antibodies that recognize the precursor forms of the growth factor. These studies were complemented by immunoblot analysis of proTGF alpha in separated uterine cell-type preparations. The specificity of the antibodies used in these studies was confirmed by use of pancreas or lactating mammary glands from transgenic mice in which mutated proTGF alpha, lacking recognition sites for proteolytic cleavages, was targeted for expression under a tissue-specific enhancer/promoter. Analysis of histochemical studies revealed accumulation of immunoreactive proTGF alpha primarily in luminal and glandular epithelial cells on Day 1 of pregnancy or pseudopregnancy followed by little or no accumulation on Days 2 and 3. However, immunoreactive proTGF alpha started to reappear in the luminal epithelium on the morning of Day 4 and became more prominent in the afternoon. In pregnant mice, immunostaining persisted in these cells at the implantation sites during the time of attachment reaction (2130 h on Day 4), but disappeared by morning of Day 5. Immunostaining appeared to be situated at the apical border of the luminal epithelium. No positive immunostaining could be detected in the nonreceptive uterus on Day 5 or 6 of pseudopregnancy. Consistent with the immunohistochemistry results, Western blot analysis detected two species of precursor proteins (14.5 and 17 kDa) in isolated luminal epithelial cell-enriched preparations on Day 4, but not on Day 5, of pseudopregnancy. The results suggest that proTGF alpha accumulates in the luminal epithelium of the receptive uterus prior to implantation. The effects of ovarian steroids on uterine accumulation of proTGF alpha were examined in ovariectomized adult mice by immunohistochemistry and immunoblotting. Whereas an injection of estradiol-17 beta (E2) or progesterone (P4) had little or a modest effect on epithelial accumulation of proTGF alpha, P4 priming for several days resulted in distinct accumulation of proTGF alpha in epithelial cells. The superimposition of an E2 treatment on P4 priming showed a biphasic response, with an initial gradual loss of immunostaining through 12 h followed by a return by 24 h of E2 treatment. The combined hormone treatment schedule employed here is similar to the situation of inducing implantation with E2 in P4-primed delayed implanting mice. The results suggest a paracrine/"juxtacrine" role for this growth factor in implantation.

A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon.

EDA is a facultative type III homology of human fibronectin encoded by an alternative spliced exon. The EDA+ and EDA- mRNA forms show a cell type specific distribution with their relative proportion varying during development, aging and oncogenic transformation. We have previously demonstrated that an 81 bp nucleotide sequence within the exon itself is essential for differential RNA processing. Fine mapping of cis acting elements within this region has been carried out to identify possible target sites for the modulation of alternative splicing. There are at least two short nucleotide sequences involved. Element A (GAAGAAGA) is a positive modulator for the recognition of the exon, its deletion results in constitutive exclusion of the EDA exon. Element B (CAAGG) is a negative modulator for exon recognition, its deletion results in constitutive inclusion of the EDA exon. This bipartite structure of the splicing enhancer is a novel feature of the mammalian exons.

Lysosomal integral membrane proteins exhibit region and cell type specific distribution in the epididymis of the adult rat.

The epididymis, a post-testicular site required for maturation and storage of spermatozoa, is actively involved in exocytic and endocytic events, two phenomena likely to depend on the integrity of the lysosomal system. To study the lysosomal system of the epididymis, five monoclonal antibodies, previously characterized as recognizing five distinct lysosomal integral membrane proteins (LIMPs 1-5), were used as molecular probes of lysosome distribution in cells lining the epithelium. Immunocytochemical localization of LIMPs, using biotin-streptavidin immunoperoxidase methodology, was performed on frozen sections of adult rat epididymides and in cell cultures prepared from either the caput or cauda epididymis. In frozen sections, a heterogeneous distribution of the different LIMPs along the length of the epididymis was observed. For example, the distribution of LIMP 1 (35-50 K) was detected in all cells of the caput and quite dramatically in clear cells of the distal caput, corpus, and cauda epididymis, but specifically not in the principal cells of the distal caput, corpus, and cauda. In contrast, LIMP 2 (64-71 K) was present in all cells of the epididymis, except clear cells. LIMPs 4 and 5 (93 K and 93 K) were detected in all epididymal cells, including the clear cells. Finally, whereas the regional and cell type distribution of LIMP 3 (74 K) in the epididymis was identical to that of LIMPs 4 and 5, the nature of the vesicles immunostained was distinct. In cultured cells, the general immunostaining patterns observed in vivo were maintained during the duration of the primary cultures for all five LIMPs. Our results begin to address the molecular heterogeneity of the lysosomal system along the length of the epididymis, and may suggest in part a basis for underlying structural and functional characteristics of the epididymis leading to the sequential maturation of sperm.

Annexins in cardiac tissue: cellular localization and effect on phospholipase activity.

Stimulation of cardiac phospholipid metabolism has diverse biological effects, ranging from subtle changes in cellular function to severe cellular damage. Accordingly, knowledge of the factors governing the activity of cardiac phospholipases is of great biological importance. A possible role of annexins, intracellular proteins that bind to membranes in a calcium dependent manner, as modulators of phospholipase activity has been proposed. In this study we investigated the cell type specific distribution of Annexin V and VIII in the heart. Recombinant Annexin V was used to examine the effect of this type of Annexin on cardiac phospholipase activity. Western blot analysis shows that annexin V is abundantly present in the heart. Using isolated myocytes and cultured cardiac endothelial and fibroblast-like cells, it is demonstrated that the localization of Annexin V is confined to non-myocytes. No positive bands matching the Mw of recombinant Annexin VIII are found in any of the cell types examined. In vitro studies demonstrate that recombinant Annexin V potently inhibits the activity of cardiac membrane-bound phospholipases, acting on their natural surrounding substrate, in a calcium dependent manner. Interestingly, annexin V also inhibits triacylglycerol hydrolysis. In conclusion, the expression of annexins is cell-type specific and suggests a cell-type specific function of these proteins in the heart. The absence of Annexin V in cardiac myocytes dismisses involvement of this annexin in cardiomyocyte phospholipid metabolism. The presence of Annexin V in cardiac endothelial and fibroblasts suggests a regulating role in the phospholipid homeostasis of non-myocyte cell types in the heart.

Differential regional expression of three natriuretic peptide receptor genes within primate tissues.

The natriuretic peptide receptors are three homologous cell surface proteins, each with a single transmembrane domain. The atrial natriuretic peptide receptor type A (ANPRA) and the homologous receptor type B (ANPRB) are both membrane guanylyl cyclases that synthesize cyclic GMP as an intracellular second messenger. The third receptor in this family, the atrial natriuretic peptide receptor type C (ANPRC), is not coupled to cyclic GMP production. We report on the distribution of the ANPRA, ANPRB, and ANPRC mRNAs in rhesus monkey tissues assayed by in situ hybridization. ANPRA mRNA is most abundantly expressed in the kidney glomerulus, adrenal zona glomerulosa, pituitary, cerebellum, and endocardial endothelial cells of the right and left atrium and right ventricle. In contrast, abundant ANPRB expression appears to be confined to the adrenal medulla, pituitary, and cerebellum. ANPRC mRNA appeared to be expressed very differently than ANPRA and ANPRB. In the heart, ANPRC mRNA is expressed most prominently in endocardial endothelial cells of all four chambers but is also found throughout the myocardium only in the right atrium. These data identify major sites of natriuretic peptide receptor mRNA expression and suggest that there may be prominent cell type-specific differential distribution of these receptors in central and peripheral targets for the natriuretic peptides.

Immunocytochemical visualization of muscarinic cholinoceptors in the human cerebral cortex

The monoclonal antibody M 35 was used to study the cellular and subcellular distribution of muscarinic acetylcholine receptors in the human cerebral cortex. M 35, raised against purified muscarinic receptor protein, exerts muscarinic agonist-like effects at cholinergic synapses. Applied to human cortical tissue immediately fixed upon neurosurgical removal, light microscopically M 35 revealed immunoreactive perikarya predominantly of the pyramidal cell type in layers II/III and V, their labeled apical dendrites extending into the superficial layers. Furthermore, a smaller number of neurons of various non-pyramidal morphology in layers VI, IV and III was immunopositive. At the ultrastructural level, immunoprecipitate decorated distinct regions of the perikaryal cytoplasm, numerous dendritic profiles and synapses of both symmetric and asymmetric appearance. In the perikarya immunoprecipitate was associated with ribosomes, the endoplasmic reticulum and the Golgi apparatus. In dendrites the microtubular system, in synaptic complexes the postsynaptic membranes were decorated. The results suggest that novel informations about the cell type specific and subcellular distribution of human muscarinic cholinoceptors can be obtained by M 35 immunocytochemistry.

Immunocytogenetics

We report a nonhistone antigen to be cell type-specifically associated with constitutive heterochromatin. Human autoantibodies were used to analyze by indirect immunofluorescence the pattern of association of the antigenic protein with the heterochromatin of murine chromosomes, as well as those of other representative vertebrate species. The evolutionary stability of its cell type-specific distribution pattern suggests that this nonhistone antigen plays an important role in the structure and/or function of constitutive heterochromatin. In mitotic chromosomes, the antigen was localized to discrete granules scattered throughout the entire chromatin. These structural elements may function as condensation centers, with each granule representing an aggregation of anchoring complexes for the chromatin loops.

Cell type-specific distribution of cathepsin B and D immunoreactivity within the rabbit retina.

The cellular localization of cathepsin B and D immunoreactivity was demonstrated at the light microscopic level in the retina of adult rabbits by use of the peroxidase-antiperoxidase technique. Antisera were raised against rat liver enzymes. Whereas cathepsin D immunoreactivity was confined to Müller (glial) cells, cathepsin B was demonstrated in some, but not all, neuronal cell types. It is proposed that the two enzymes might carry different functions within the neuronal versus glial compartment.