• Intercellular Communication by Exchange of Cytoplasmic Material via Tunneling Nano-Tube Like Structures in Primary Human Renal Epithelial Cells

      Domhan, Sophie; Ma, Lili; Tai, Albert; Anaya, Zachary; Beheshti, Afshin; Zeier, Martin; Hlatky, Lynn; Abdollahi, Amir; McNeil, Paul L.; Department of Cellular Biology and Anatomy; et al. (2011-06-27)
      Transfer of cellular material via tunneling nanotubes (TNT) was recently discovered as a novel mechanism for intercellular communication. The role of intercellular exchange in communication of renal epithelium is not known. Here we report extensive spontaneous intercellular exchange of cargo vesicles and organelles between primary human proximal tubular epithelial cells (RPTEC). Cells were labeled with two different quantum dot nanocrystals (Qtracker 605 or 525) and intercellular exchange was quantified by high-throughput fluorescence imaging and FACS analysis. In co-culture, a substantial fraction of cells (67.5%) contained both dyes indicating high levels of spontaneous intercellular exchange in RPTEC. The double positive cells could be divided into three categories based on the preponderance of 605 Qtracker (46.30%), 525 Qtracker (48.3%) and approximately equal content of both Qtrackers (4.57%). The transfer of mitochondria between RPTECs was also detected using an organelle specific dye. Inhibition of TNT genesis by actin polymerization inhibitor (Latrunculin B) markedly reduced intercellular exchange (>60%) suggesting that intercellular exchange in RPTEC was in part mediated via TNT-like structures. In contrast, induction of cellular stress by Zeocin treatment increased tube-genesis in RPTEC. Our data indicates an unexpected dynamic of intercellular communication between RPTEC by exchange of cytosolic material, which may play an important role in renal physiology.
    • Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

      Ariga, Junko; Walker, Steven L.; Mumm, Jeff S.; Department of Cellular Biology and Anatomy (2010-09-20)
      High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
    • Male accessory gland protein reduces egg laying in a simultaneous hermaphrodite.

      Koene, Joris M; Sloot, Wiebe; Montagne-Wajer, Kora; Cummins, Scott F; Degnan, Bernard M; Smith, John S; Nagle, Gregg T; ter Maat, Andries; Department of Cellular Biology and Anatomy (2010-04-20)
      Seminal fluid is an important part of the ejaculate of internally fertilizing animals. This fluid contains substances that nourish and activate sperm for successful fertilization. Additionally, it contains components that influence female physiology to further enhance fertilization success of the sperm donor, possibly beyond the recipient's optimum. Although evidence for such substances abounds, few studies have unraveled their identities, and focus has been exclusively on separate-sex species. We present the first detailed study into the seminal fluid composition of a hermaphrodite (Lymnaea stagnalis). Eight novel peptides and proteins were identified from the seminal-fluid-producing prostate gland and tested for effects on oviposition, hatching and consumption. The gene for the protein found to suppress egg mass production, Ovipostatin, was sequenced, thereby providing the first fully-characterized seminal fluid substance in a simultaneous hermaphrodite. Thus, seminal fluid peptides and proteins have evolved and can play a crucial role in sexual selection even when the sexes are combined.
    • Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart.

      Maddox, Dennis M; Manlapat, Anna K; Roon, Penny; Prasad, Puttur D; Ganapathy, Vadivel; Smith, Sylvia B; Department of Cellular Biology and Anatomy; Department of Obstetrics and Gynecology; Department of Biochemistry and Molecular Biology; Department of Ophthalmology (2003-10-29)
      BACKGROUND: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively. RESULTS: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE). CONCLUSIONS: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.
    • Loss, Restoration, and Maintenance of Plasma Membrane Integrity

      McNeil, Paul L.; Steinhardt, Richard A.; Department of Cellular Biology and Anatomy (1997-04-7)

      Black, Owen; Bresnick, Edward; Department of Cellular Biology and Anatomy (1972-03-1)
      The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, 14C-labeled amino acids and δ-aminolevulinic acid-3H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-14C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b5, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b5 region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.

      Cantrell, Elroy; Bresnick, Edward; Department of Cellular Biology and Anatomy (1972-02-1)
      Previous studies have implicated the reticuloendothelial cells of the liver in certain aspects of steroid metabolism. The similarity in the metabolism of steroids and polycyclic hydrocarbons suggested that the nonparenchymal cells possibly play a role in these areas . The present study presents evidence that at least one of the microsomal NADPH-requirig enzymes, benzpyrene hydroxylase, is present in nonparenchymal cells and, furthermore, is "inducible ." In adult rats treated with 3-methylcholanthrene or ß-naphthoflavone, the nonparenchymal cells exhibited increases in benzpyrene hydroxylase activity of 17-fold and five-fold, respectively . Treatment with phenobarbital resulted in only a slight increase in enzyme activity . Enzyme activity in parenchymal cells under similar conditions was increased sixfold and fivefold by 3-methylcholanthrene and ß-naphthoflavone, respectively, but not by phenobarbital.