The Pennsylvania State University The Graduate School Eberly College of Science A SYSTEMATIC STUDY OF EXPRESSION AND FUNCTION OF CAENORHABDITIS ELEGANS GENES ENCODING P-TYPE ATPASES IN SUBFAMILY IV, THE GROUP OF PUTATIVE TRANSBILAYER AMPHIPATH TRANSPORTERS A Thesis in Intergative Biosciences by Nicholas N. Lyssenko © 2007 Nicholas N. Lyssenko Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2007 The thesis of Nicholas N. Lyssenko was reviewed and approved * by the following: Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology Thesis Adviser Chair of the Committee Simon Gilroy Professor of Biology Andrea M. Mastro Professor of Microbiology and Cell Biology Wendy Hanna-Rose Assistant Professor of Biochemistry and Molecular Biology Graham H. Thomas Associate Professor of Biology and of Biochemistry and Molecular Biology * Signatures are on file in the Graduate School. ABSTRACT In eukaryotic organisms, the cytofacial and exofacial monolayers of the plasma membrane and certain other cell membranes exhibit substantial differences with respect to lipid composition. The plasma membrane usually contains all of its phosphatidylserine (PS) and a larger portion of phosphatidylethanolamine in the inner leaflet. Viable cells normally conceal plasma membrane PS, while cells undergoing apoptosis or necrosis expose it on the cell surface. P-type ATPases in subfamily IV are the leading candidate transbilayer amphipath transporters (TATs), the proteins postulated to actively translocate PS to the cytofacial half of the compositionally polarized membranes. TATs of the single cell fungus Saccharomyces sereviciae are presently the best studied. Several mammalian members of the group are also under investigation. However, the currently available data fail to provide a comprehensive view of tissue-specific expression and developmental function of the subfamily members in a multicellular organism. Here are reported findings of the study aimed to systematically investigate subfamily IV P-type ATPases of the nematode Caenorhabditis elegans. The C. elegans genome contains six open reading frames that encode P-type ATPases in subfamily IV. All six are transcribed. Expression of a nucleus targeted green fluorescent protein reporter under the control of tat-2, tat-3, tat-4 or tat-5 promoter sequence showed that the former three genes are active in tissue-specific patterns, while the latter gene is transcribed ubiquitously. Phenotypic analysis of nematodes with temporarily, via RNA interference, or permanently, by deletion mutagenesis, altered expression of tat-1 through 5 shows that tat-5 is an essential gene. tat-5(RNAi) animals exhibit morphological defects of the gonad, fertilization deficiencies and embryonic lethality. tat-1 through 4 are nonessential. The tat-3; tat-4 gene pair is nonessential as well. tat-1 seems to be required for exposure of PS on the surface of apoptotic cells in the nematode gonad. tat-2 and tat-4 appear to mediate cholesterol metabolism in the digestive system. No notable phenotype was observed with tat-3. These findings from C. elegans suggest that tat-1 through 4 are tissue specific genes that regulate membrane structure, and tat-5 is the sole housekeeping TAT. iii TABLE OF CONTENTS List of figures…………………………………………………………………………… vii List of tables……………………………………………………………………………. ix List of abbreviations…………………………………………………………………… x Acknowledgements..…………………………………………………………………... xii Chapter 1. A brief overview of the research project and the organization of this 1 thesis Chapter 2. An introduction to cell membranes, membrane phospholipid asymmetry and transbilayer lipid transport……………………………………......... 4 Cell membrane as a two dimensional liquid………………………………... 4 Asymmetric lipid distribution between the two monolayers of certain cell membranes…………………………………………………………………….... 17 Phospholipid transbilayer flip-flop……………………………………………... 20 P-type ATPases in subfamily IV as putative transbilayer amphipath transporters........…………………………………………………………........ 29 Caenorhabditis elegans as a model system for investigations of lipid bilayers…………………………………………………………………………... 32 References………………………………………………………………………. 34 Chapter 3. A brief review of the origins and major developments of the plasma membrane hypothesis………………………………………………………………… 48 Plasma membrane as the problem of permeability and osmosis………….. 48 Plasma membrane as the problems of cell electrical resistance and cell surface area……………………………………………………………………... 57 Early direct observations of cell membranes………………………………… 70 References………………………………………………………………………. 75 Chapter 4. Structure and transcription of tat-1 through 5 genetic loci in C. elegans………………………………………………………………………………….. 83 Introduction………………………………………………………………………. 83 Materials and methods…………………………………………………………. 86 mRNA isolation and cDNA synthesis…………………………………. 86 Rapid amplification of cDNA ends (RACE)………………………….. 87 EST sequencing………………………………………………………… 89 Results…………………………………………………………………………… 89 tat-1………………………………………………………………………. 89 tat-2 and tat-3……………………………………………………………. 93 tat-4 and T24H7.6………………………………………………………. 97 tat-5 and tat-6……………………………………………………………. 104 Discussion ………………………………………………………………………. 104 iv References………………………………………………………………………. 109 Chapter 5. Expression patterns of the GFP reporter under the control of tat-2, 3, 4 or 5 promoter sequences………………………………………………………… 111 Introduction……………………………………………………………………… 111 Materials and methods………………………………………………………..... 115 YAC DNA isolation……………………………………………………… 115 Cosmid DNA isolation………………………………………………….. 116 Expression cassette construction…………………………………….. 116 tat-1………………………………………………………………. 116 tat-2………………………………………………………………. 118 tat-3………………………………………………………………. 120 tat-4………………………………………………………………. 120 tat-5………………………………………………………………. 124 Particle bombardment delivery of the expression cassettes into the nematode gonad………………………………………………………… 124 Image acquisition and processing…………………………………….. 128 Results…………………………………………………………………………… 128 Biolistic transformation with tat expression cassettes that include a large portion of the 5’ translated sequence failed to produce stable GFP-fluorescent transgenic lines……………………………………… 128 tat-2 is expressed in the alimentary, reproductive and excretory tissues……………………………………………………………………. 133 tat-3 is expressed in the alimentary, epithelial and reproductive systems………………………………………………………………….. 139 tat-4 is expressed in the alimentary and reproductive systems……. 144 tat-5 is expressed broadly in many tissues………………………….. 148 Discussion……………………………………………………………………….. 150 Expression patterns of tat-2 and tat-4 are similar and distinct from the expression pattern of tat-3………………………………………… 150 Coordinated expression of tat-2 and tat-3 in the developing vulva and the uterine-vulval connection…………………………………….. 152 Expression and localization homology between TAT-2 and its mammalian counterparts………………………………………………. 154 tat-5 is the most broadly expressed of the 4 investigated tats…….. 155 References………………………………………………………………………. 156 Chapter 6. Phenotypic analysis of Caenorhabditis elegans animals with altered expression of tat-1, 2, 3, 4 or 5………………………………………………………. 159 Introduction………………………………………………………………………. 159 Materials and methods…………………………………………………………. 163 C. elegans mutants and transgenes and animal maintenance…….. 163 tat RNAi vectors…………………………………………………………. 165 dsRNA feeding of nematodes to induces RNAi……………………… 165 v The assay for PS expose on the surface of apoptotic germ cells…. 166 Sterol deprivation assay………………………………………………... 167 Results…………………………………………………………………………… 168 tat-5 is an essential gene………………………………………………. 168 tat-1, tat-2, tat-3 and tat-4 are nonessential and not required for apoptotic cell clearance………………………………………………… 170 Apoptotic germline cell corpse staining with GFP-Annexin V is compromised in tat-1(RNAi) animals…………………………………. 170 tat-2 and tat-4 mediation of sterol metabolism……………………… 174 tat-3; tat-4 double mutant is viable……………………………………. 180 Discussion……………………………………………………………………….. 183 tat-5 might be a housekeeping gene………………………………….. 183 Involvement of the putative APLTs in sterol metabolism…………… 183 Lipid asymmetry and TATs…..………………………………………… 189 Future research directions……………………………………………... 191 References………………………………………………………………………. 192 vi LIST OF FIGURES Chapter 2. Figure 1. Chemical structure of a representative phospholipid, phosphatidylcholine...………………………………………………………………..... 6 Figure 2. X-ray diffraction crystal structures of dilauroyl phosphatidylethanolamine and dimyristoyl phosphatidylcholine....………………. 8 Figure 3. Lipid phase behavior……………………………………………………….. 10 Figure 4. Lipid translocation in the mammalian plasma membrane……………… 28 Chapter 3. Figure 1. Electrical properties of cell suspensions and tissues…………………… 60 Figure 2. The Langmuir trough and Langmuir monomolecular films…………….. 62 Figure 3. Most influential models of the cell membrane…………………………... 64 Figure 4. A schematic of the surface of the oil droplet from the mackerel egg…. 67 Figure 5. An electron density profile, and its interpretation, of rabbit sciatic myelin…………………………………………………………………………………… 71 Figure 6. The unit membrane………………………………………………………… 72 Chapter 4. Figure 1. Amplification of tat-1, 2 and 3 cDNA transcripts with a gene-specific and SL1-specific primers……………………………………………………………… 90 Figure 2. Structure and transcription of the tat-1 locus……………………………. 91 Figure 3. Structure and expression of the tat-2 locus……………………………… 94 Figure 4.