NUCLEAR ENVELOPE DYNAMICS IN EMBRYOS AND SOMATIC CELLS

By DANIEL LEE

CONTENTS 1 DYNAMICS OF THE VERTEBRATE NUCLEAR ENVELOPE 1 ABSTRACT 1 INTERPHASE NUCLEAR ENVELOPE STRUCTURE 1 NUCLEAR ENVELOPE DISASSEMBLY 3 NUCLEAR ASSEMBLY 5 CONCLUDING REMARKS 8 2 DYNAMICS OF NUCLEAR ENVELOPE PROTEINS DURING THE CELL CYCLE IN MAMMALIAN CELLS 15 ABSTRACT 15 WHY SHOULD NUCLEAR ENVELOPE PROTEINS BE DYNAMIC 15 WHAT IS THE NUCLEAR ENVELOPE MADE OF 16 STUDYING NUCLEAR ENVELOPE PROTEIN DYNAMICS 17 DYNAMICS IN INTERPHASE 17 CHROMOSOMES DO NOT MOVE MUCH IN INTERPHASE 21 DYNAMICS IN MITOSIS 21 INM PROTEINS SWITCHING RETENTION OFF AND BACK ON 21 LAMINA TEARING OF A POLYMER DISPERSION AND RE IMPORT OF MONOMERS 23 PORE COMPLEX DISASSEMBLY AND ASSEMBLY MANY OPEN QUESTIONS 24 CHROMOSOMES A COMPLEX TEMPLATE FOR NUCLEAR ASSEMBLY 25 CONCLUDING REMARKS 25 3 TARGETING AND RETENTION OF PROTEINS IN THE INNER AND PORE MEMBRANES OF THE NUCLEAR ENVELOPE 29 ABSTRACT 29 TARGETING OF INTEGRAL MEMBRANE PROTEINS TO THE INNER NUCLEAR MEMBRANE 29 TARGETING AND RETENTION OF INTEGRAL MEMBRANE PROTEINS TO THE PORE MEMBRANE 35 TARGETING OF PERIPHERAL MEMBRANE PROTEINS TO THE INNER NUCLEAR MEMBRANE 36 CONCLUSION 38 4 DYNAMIC CONNECTIONS OF NUCLEAR ENVELOPE PROTEINS TO CHROMATIN AND THE NUCLEAR MATRIX 43 ABSTRACT 43 INTRODUCTION 43 MAJOR COMPONENTS OF THE PERIPHERAL NUCLEAR LAMINA 44 LAMINA PROTEINS IN THE NUCLEAR INTERIOR 46 INTERACTIONS AT THE INTERFACE BETWEEN THE LAMINA AND THE NUCLEAR SCAFFOLD CHROMATIN 47 POTENTIAL FUNCTIONS OF LAMINA PROTEINS IN INTERPHASE 49 DYNAMICS AND FUNCTIONS OF LAMINA CHROMATIN INTERACTIONS DURING MITOSIS 50 CONCLUSIONS AND FUTURE PROSPECTS 53 5 ROLE OF RAN GTPASE IN NUCLEAR ENVELOPE ASSEMBLY 61 ABSTRACT 61 BACKGROUND 61 CONTROL OF NUCLEAR ENVELOPE ASSEMBLY BY RAN 64 6 MITOTIC CONTROL OF NUCLEAR PORE COMPLEX ASSEMBLY 73 INTRODUCTION 73 THE NUCLEAR LAMINA 73 THE INNER NUCLEAR MEMBRANE 73 NUCLEAR PORE COMPLEXES 74 DYNAMICS OF THE NUCLEAR ENVELOPE DURING MITOSIS 76 NUCLEAR ENVELOPE BREAKDOWN 76 NUCLEAR ENVELOPE REFORMATION 77 NPC ASSEMBLY 77 WHEN DOES THE NPC BECOME FUNCTIONAL 81 SUMMARY 82 7 STRUCTURE FUNCTION AND BIOGENESIS OF THE NUCLEAR ENVELOPE IN THE YEAST SACCHAROMYCES CEREVISIAE 87 INTRODUCTION 87 OVERVIEW OF THE YEAST NPC AND ITS FUNCTION IN TRANSPORT 88 COMPOSITION AND STRUCTURE FUNCTION RELATIONSHIPS OF THE YEAST NPC 89 BIOGENESIS OF THE YEAST NPCS AND THEIR ROLE IN THE ORGANIZATION OF THE NE 93 INTEGRAL MEMBRANE PROTEINS OF THE YEAST NE AND THEIR FUNCTION 96 8 NUCLEAR ENVELOPE BREAKDOWN AND REASSEMBLY IN C ELEGANS EVOLUTIONARY ASPECTS OF LAMINA STRUCTURE AND FUNCTION 103 ABSTRACT 103 THE STRUCTURE AND PROTEIN COMPOSITION OF THE NUCLEAR LAMINA IN C ELEGANS 103 POSSIBLE FUNCTIONS OF THE NUCLEAR LAMINA IN C ELEGANS 104 NUCLEAR DYNAMICS IN C ELEGANS DURING MITOSIS 106 9 NUCLEAR ENVELOPE ASSEMBLY IN GAMETES AND PRONUCLEI 111 ABSTRACT 111 INTRODUCTION 111 BACKGROUND 111 SPERM NUCLEAR ENVELOPE DISASSEMBLY 112 MEMBRANE VESICLE FRACTIONS CONTRIBUTING TO THE NUCLEAR ENVELOPE 113 BINDING OF EGG CYTOPLASMIC VESICLES TO SPERM CHROMATIN AND NUCLEAR ENVELOPE REMNANTS 115 FUSION OF NUCLEAR ENVELOPE PRECURSOR VESICLES 117 COMPLETION OF MALE PRONUCLEAR ENVELOPE FORMATION 123 COMPARISON WITH OTHER SYSTEMS AND SPECULATIONS 123 ISSUES FOR FUTURE INVESTIGATION 127 10 NUCLEAR ENVELOPE DYNAMICS IN DROSOPHILA PRONUCLEAR FORMATION AND IN EMBRYOS 131 DROSOPHILA NUCLEAR ENVELOPES 131 DEVELOPMENTAL CHANGES IN NUCLEAR ENVELOPES AROUND THE TIME OF FERTILIZATION 132 CONCLUSION 138 11 THE DISTRIBUTION OF EMERIN AND LAMINS IN X LINKED EMERY DREIFUSS MUSCULAR DYSTROPHY 143 INTRODUCTION 143 A BRIEF HISTORY OF EDMD 143 THE NORMAL DISTRIBUTION OF EMERIN AND LAMINS 145 DISTRIBUTION OF EMERIN AND LAMINS IN X LINKED EDMD 148 12 LAMINOPATHIES ONE GENE TWO PROTEINS FIVE DISEASES 153 ABSTRACT 153 INTRODUCTION 153 DISORDERS OF CARDIAC AND OR SKELETAL MUSCLES LINKED TO LMNA ALTERATIONS 154 LIPODYSTROPHIES AND THE FAMILIAL PARTIAL LIPODYSTROPHY OF THE DUNNIGAN TYPE FPLD 159 FAMILIAL PARTIAL LIPODYSTROPHY OF THE DUNNIGAN TYPE FPLD 162 COULD SOME PATIENTS WITH LMNA MUTATIONS BE AFFECTED BY BOTH SKELETAL OR CARDIAC MUSCULAR SYMPTOMS AND LIPODYSTROPHY 163 EXPERIMENTAL MODELS OF LAMIN A C ALTERATIONS 163 NUCLEAR ALTERATIONS IN CELLS HARBORING LMNA MUTATIONS 164 CONCLUSION 166 ADDENDUM 167 CHAPTER 1 DYNAMICS OF THE VERTEBRATE NUCLEAR ENVELOPE ABSTRACT T HE CELL NUCLEUS IS A COMPLICATED ORGANELLE THAT HOUSES THE GENOME OF HUMANS AND OTHER EUKARYOTIC ORGANISMS CHROMOSOMES ARE ENCLOSED BY THE NUCLEAR ENVELOPE AND COMMUNICATE WITH THE CYTOPLASM BY THE REGULATED MOVEMENT OF MOLECULES ACROSS NUCLEAR PORE COMPLEXES IN MULTICELLULAR ANIMAL EUKARYOTES METAZOANS A SPECIAL SET OF NUCLEAR MEMBRANE PROTEINS AND LAMIN FILAMENTS INTERACT WITH CHROMATIN TO PROVIDE KEY STRUCTURAL AND FUNCTIONAL ELEMENTS TO THE NUCLEUS REMARKABLY THESE STRUCTURES ARE REVERSIBLY DISASSEMBLED DURING MITOSIS THIS CHAPTER DESCRIBES THE STRUCTURE AND MAJOR CONSTITUENT PROTEINS OF THE METAZOAN NUCLEAR ENVELOPE OUR CURRENT UNDERSTANDING OF NUCLEAR ENVELOPE DYNAMICS DURING MITOSIS AND PATHWAYS FOR THE REVERSIBLE BREAKDOWN AND REASSEMBLY OF THE NUCLEAR ENVELOPE AND NUCLEAR INFRASTRUCTURE THIS FIELD IS MOVING QUITE FAST A BETTER UNDERSTANDING OF THESE FUNDAMENTAL ASPECTS OF NUCLEAR ENVELOPE STRUCTURE AND DYNAMICS WILL PROVIDE NEW INSIGHTS INTO AN EMERGING CLASS OF INHERITED HUMAN DISEASES INCLUDING EMERY DREIFUSS MUSCULAR DYSTROPHY DILATED CARDIOMYOPATHY AND LIPODYSTROPHY FURTHER WORK IN THIS FIELD MAY ALSO SUGGEST NOVEL ANTI VIRAL THERAPIES FOR HIV OR HERPESVIRUS WHICH SPECIFICALLY DISRUPT NUCLEAR ENVELOPE STRUC TURE DURING THEIR LIFE CYCLES INTERPHASE NUCLEAR ENVELOPE STRUCTURE THE NUCLEUS OF METAZOAN CELLS INCLUDES HIGHLY STABLE STRUCTURES SUCH AS THE CHROMOSOMES THE NUCLEAR ENVELOPE AND LAMINA PLUS HIGHLY MOBILE PROTEINS RESPONSIBLE FOR RNA PRODUCTION AND NUCLEAR METABOLISM THIS COMPLEX ARCHITECTURE IS REVERSIBLY DISASSEMBLED DURING MITOSIS IN THIS CHAPTER WE SUMMARIZE INTERPHASE NUCLEAR STRUCTURE AND THE EVENTS AND MECHANISMS OF NUCLEAR ENVELOPE DISASSEMBLY AND REASSEMBLY DURING MITOSIS THE NUCLEAR ENVELOPE DEFINES AND ENCLOSES THE CELL NUCLEUS THE ENVELOPE IS COMPOSED OF TWO CONCENTRIC MEMBRANES OUTER AND INNER AND NUCLEAR PORE COMPLEXES THAT ARE ANCHORED BY A NETWORK OF FILAMENTS TERMED THE NUCLEAR LAMINA THE OUTER MEMBRANE IS CONTINUOUS WITH AND HAS THE SAME PROTEIN COMPOSITION AS THE ROUGH ENDOPLASMIC RETICULUM ER THE OUTER AND INNER NUCLEAR MEMBRANES FUSE PERIODICALLY TO FORM NUCLEAR PORES PORES HAVE A DIAMETER OF 100 NM AND ARE OCCUPIED BY NUCLEAR PORE COMPLEXES NUCLEAR PORE COMPLEXES ACTIVELY MEDI ATE THE TRANSPORT OF MACROMOLECULES BETWEEN THE NUCLEUS AND THE CYTOPLASM THEY ALSO PROVIDE AQUEOUS CHANNELS THROUGH WHICH IONS AND SMALL PROTEINS 40 60 KD CAN DIFFUSE PASSIVELY 1 UNLIKE THE NUCLEAR OUTER MEMBRANE THE INNER MEMBRANE HAS A UNIQUE PROTEIN COMPOSITION AND CAN THUS BE VIEWED AS A HIGHLY SPECIALIZED SUBDOMAIN OF THE ER PROTEINS UNIQUE TO THE NUCLEAR INNER MEMBRANE INCLUDE THE LAMIN B RECEPTOR LBR 2 SEVERAL ISOFORMS EACH OF THE LAMINA ASSOCIATED POLYPEPTIDES LAPS 1 AND 2 3 5 EMERIN 6 7 MAN1 8 9 NURIM10 AND THE RING FINGER BINDING PROTEIN RFBP 11 MANY OF THESE PROTEINS CAN BIND DIRECTLY TO NUCLEAR LAMINS WHICH ARE ABUNDANT NEAR THE INNER MEMBRANE WE WILL REFER TO THE LAMIN 1