Immunofluorescence microchamber technique for characterizing liver cell organelles in vitro

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AASLD ABSTRACTS

HEPATOLOGY October 2001

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USE OF DIFFERENT DYNAMIN FORMS IN D I S T I N C T HEPATOCELLUEAR ENDOCYTIC PROCESSES. Hong Cao, Heather M Thompson, Bing Q

MICROTUBULAR-DEPENDENT BIDIRECTIONAL TRAFFICKING OF BSEP IN POLARIZED WIF-B9 CELLS. Yoshiyuki Wakabayashi, Irwin M

Huang, Mark A McNiven, Mayo Clinic, Rochester, MN

Arias, Dept of Physiology, Tufts Univ Sch of Medicine, Boston, MA

The large GTPase dynamin is a mechanoenzyme that mediates the scission of nascent clathrin-coated as well as nonclathrin-coated vesicles from the plasma membrane (PM) during endocytosis. So far, three isoforms of dynamin have been identified, each containing multiple splice variants resulting in at least 25 different dynamin proteins from the three genes. In earlier studies we have shown there is a differential distribution of three dynamin 2 (Dyn 2) splice variants in hepatocytes Dyn 2(aa), (ab), and (ba). All three of these Dyn 2 splice variants are localized to numerous punctate spots on the plasma membrane (PM) with Dyn 2(aa) and (ba) showing a perinuclear distribution as well (Cao et al., MBC 9: 2595). Currently, it is unclear whether these different Dyn 2 splice variants perform distinct or redundant cellular functions. Therefore, the GOAL of this study was to analyze the distribution and endocytic functions of these three Dyn 2 splice forms in hepatocytes using the K44A GTPasemutant of each splice form. Mutants of each of the Dyn 2 splice variants, with and without a GFP-tag, were expressed in either rat hepatocytes (Clone 9) or rat fibroblasts (FR). Subsequently, cells were either fixed and stained for clathrin or challenged to take up an endocytic marker, then processed and viewed using a confocal microscope. RESULTS: Cells expressing non-tagged Dyn 2(aa) K44A displayed not only short tubules at the PM, but also a perinuclear tubular network. Cells expressing non-tagged as well as GFP-tagged Dyn 2(ab) K44A exhibited extensive PM tubules, similar to those seen in shibire Garland ceils, which appeared to be pulled towards the center of the cell. Lastly, cells expressing non-tagged or GFP-tagged Dyn 2(ba) K44A showed a diffuse cytoplasmic localization as well as disordered PM and perinuclear punctate spots. C]athrin distribution was disrupted by all three Dyn 2 mutants such that there was no longer an even PM distribution or a prominent Golgi localization. Interestingly, endocytosis of different markers was differentially inhibited by the three Dyn 2 K44A mutants. All three Dyn 2 mutants inhibited the up-take of transferrin (clathrin-mediated) to some degree. However, only Dyn 2(aa) K44A significantly inhibited the up-take of cholera toxin B, a caveolae-mediated process, and only Dyn 2(ba) K44A significantly inhibited up-take of the fluid-phase marker dextran. CONCLUSIONS: This is the first study to show that GTPase-mutants of different Dyn 2 splice forms exhibit different morphological and functional phenotypes. Though all Dyn 2 GTPase-mutants tested inhibited transferrin up-take, only Dyn 2(aa) K44A leads to inhibition of cholera toxin B up-take, whereas only Dyn 2(ba) K44A inhibited dextran up-take. These results lend support to our previous hypothesis that the various dynamin splice forms perform similar membrane severing events but at distinct cellular donor compartments.

Previous studies in rat liver and WIF-B9 cells revealed that newly synthesized canalicular membrane ABC transporters traffic directly from Golgi to the bile canalicular membrane and subsequently cycle between the canalicular membrane and tab11-associated intracellular recycling endosomes. To quantify the trafficking dynamics of canalicular ABC transporters in WIF-B9 cells, we visualized fluorescence protein-tagged BSEP(BSEP-FP) using time-lapse fluorescence microscopy and image analysis techniques. Time-lapse fluorescence imaging revealed that large tubular structures are the primary vehicles for trafficking BSEP-FP from recycling endosomes to the canalicnlar membrane. These tubular structures continually budded from recyling endosomes and underwent dynamic shape changes as they moved along microtnbular tracks to the canalicular membrane. Movement was bidirectional and oscillatory at a maximal speed of 0.93mm/sec. Microtubular depolymerization disrupted the tubular structures but did not affect the appearance of recycling endosomes. Apical membrane endocytosis of BSEP-FP was also visualized. Apical membrane protein budded from the canalicular membrane as small vesicles which detached and moved along microtubules to the recycling endosomes. Vesicular movement was oscillatory at a maximal speed of 1.85mm/sec. SUMMARY: These observations suggest that bidirectional trafficking of ABC transporters between recycling endosomes and the canalicular membrane involves different microtubular-based processes which operate at different rates and are probably regulated by different motor proteins and/or cargo-associated proteins.

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IMMUNOFLUORESCENCE MICROCHAMBER TECHNIQUE FOR CHARACTERIZING LIVER CELL ORGANELLES IN VITRO. John W Murray, Eustratios Bananis, Allan W Wolkoff, Albert Einstein Coll of Medicine, Bronx, NY

NUCLEAR LOCALIZATION OF A NOVEL HEPATOCELLULAR ISOFORM OF THE CATALYTIC SUBUNIT OF PROTEIN KINASE CK2. Philip Hilgard, Tianmin Huang, Allan W Wolkoff, Richard J Stockert, Marion Bessin Liver Research Ctr, Albert Einstein Coll of Medicine, Bronx, NY

In our pursuit of understanding the motility of liver endosomes we have devised an "optical microchamber" technique that allows for rapid localization of specific proteins to subcellular components that have bound to glass. This method represents a non-destructive, small volume means of characterizing biological material using a fluorescence microscope. Methods: A disposable microscope chamber holding as little as 3 ul is constructed from glass coverslips and double stick tape. A suspension of cellular organeUes is perfused into the chamber where organelles fortuitously stick to the glass. If desired, glass coverslips are pre-treated to facilitate binding of particular constituents, such as purified microtubules. The chamber is then washed and antibodies to specific proteins are added followed by washing and addition of fluorescent secondary antibodies. Fixatives are avoided and therefore drugs or other reagents can be added to the chamber, which then serves as an optical platform for "micro-biochemistry" experimental assays. Results: Organelles from homogenized rat liver were capable of binding untreated glass microchambers. The peripherally associated membrane proteins, dynein and kinesin were visualized on specific subsets of phase-dense material bound to the surface of the coverslip. In separate experiments, preparations of nuclei as well as endocytic vesicles also bound the coversfip within the chambers. Endocytic vesicles were prepared from rat liver that had been injected in situ with Texas-red labeled asialoorsomucoid (ASOR). Simultaneous detection of ASOR, asialoglycoprotein receptor (ASGPR), and caveolin 1 (Cavl) demonstrate that ASGPR is colocalized to ASOR vesicles, whereas many of the Cavl structures have no ASOR or ASGPR. Fluorescence intensity was shown to be linear with concentration of fluorophore allowing for quantitation using the microchamber system. Dilution series revealed that endoplasmic reticulum and Golgi (as seen by marker proteins calnexin and HA lectin) bound to chambers in a predictable, hyperbolic manner and computer algorithms were developed for image quantitation. Increasing concentrations of salt did not markedly diminish binding of organelles. The capacity for performing biochemical assays on a microscopic scale within the chamber was demonstrated by the uptake of acridine orange into endocytic vesicles upon addition of ATP. The specific vacuolar H ~ ATPase inhibitor, bafilomycin, inhibited this uptake. Conclusion: Optical microchambers provide a rapid, low volume, low cost method for characterizing biological preparations containing cellular organelles. Specific proteins can be visualized via immunofluorescence, and their abundance and spatial localization can be assessed quantitatively. In addition the system is amenable to biochemical assays using optical (e.g. fluorescent) reporters.

Background: Protein Kinase CK2 (CK2, formerly referred to as casein kinase 11)is a highly conserved and ubiquitously expressed tetrameric enzyme that phosphorylates serine/threonine residues and is essential for the viability of eukaryotic cells.The tetrameric CK2 holoenzyme consists of two catalytic a subunits and two ~ subunita, which have a stabilizing and regulatory function. Both subunits are constitutively expressed in most human tissues. The two known isoforms of the catalytic subunit, CK2a and CK2a', have been reported to have distinct tissue dependent snbcellular distributions. We hypothesize that the localization of the catalytic subunit isoforms contributes to the mechanisms that govern cellular activity and substrate specificity of CK2, which are largely unknown. Recently, we described a third isoform of the catalytic subunit, designated CK2a", which in contrast to the two others, is highly expressed in liver as compared to other tissues and involved in the regulation of hepatocellular membrane protein trafficking. The AIM of this study was to determine the subcellularlocalization of CK2a" in the human hepatoma cell line HUH-7 as well as the mechanism that regulates its distribution. Methods: Subcellular localization of CK2a" was investigated by sucrose gradient fractionation of HUH-7 cell homogenates and detection of CK2a isoforms by immunoblot analysis. The localization was confirmed by immunofluorescence microscopy. Subnuclear distribution was assessed by differential salt extractionwith or without detergent (1% NP-40) of HUH-7 nuclei and the preparation of nuclear matrix, cDNAs encoding GFP-CK2a" fusion-proteins with and without the isoform specific carboxyterminus were constructed. The distribution of these fusion-proteins was investigated by fluorescent microscopy of viable transiently transfected cells. Results: lmmunoblot analysis after sucrose gradient fractionation, as well as immunofluorescent microscopy revealed that CK2a" was localized exclusively to the nucleus of HuH-7 cells. In contrast, CK2~xand CK2a' were localized to nuclear, membrane and cytosolic compartments. Within the nucleus, CK2a" as opposed to the other CK2a isoforms was found to preferentially associate with the nuclear matrix. Expression of GFP-CK2a" fusion-protein showed that nuclear localization was dependent upon the isoform specific carboxyterminus. Deletion of the carboxyterminal 32 amino acids from the CK2a" sequence (GFPCK2a "~32) resulted in redistribution of the fusion protein to both the nucleus and the cytoplasm. To further examine the mechanism by which the isoform specific carboxyterminus inhibits nuclear-cytoplasmic shuttling, possible interference of a cryptic nuclear export signal (NES), resultingin nuclear retention of unmodified CK2a", was investigated. GFP-CK2a" a32was expressed in HUH-7 cells prior to treatment with 20 ng/ml leptomyciu B (LMB), known to strongly inhibit nuclear export mediated by leucine rich NES and potentially other export signals. In contrast to the rapid nuclear accumulation of NES containing IKB, no difference between the distribution of GFP-CK2a "A32in LMB-treated and -untreated HUH-7 cells was detected by fluorescent microscopy. Conclusions: 1. Nuclear localization of CK2a" is dependent on its carboxyterminal amino acid sequence. 2. The carboxyterminus does not inhibit nuclear-cytoplasmic shuttling by interfering with a NES motif. 3. It is likely, the carboxyterminus facilitates binding of CK2a" to the nuclear matrix or a matrix associated protein.