Assembly and Localization of the Intermediate Filament Protein, Nestin Martha Jean Marvin Submitted to the Department of Biology
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Assembly and Localization of the Intermediate Filament Protein, Nestin by Martha Jean Marvin B.A., University of California, Berkeley, 1984 Submitted to the Department of Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology January, 1995 ©()Massachusetts Institute of Technology 1994 All rights reserved Signature of Authorr ---- L-f-~Cf~L-.-----·~f-------- < / Department of Biology December 20, 1994 Certified by Ronald D.G. McKay Lab Chief, Laboratory of Molecular Biology National Institute of Neurological Diseases and Stroke National Institutes of Health Accepted by Frank Solomon Professor, Biology Chair, Department Graduate Committee MA.Z. `T,,t,'.:!NSTTUTE JAN 17 995 Assembly and Localization of the Intermediate Filament Protein, Nestin by Martha Jean Marvin Submitted to the Department of Biology on December 20, 1994 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology Abstract Neuroepithelial stem cells can be identified by their expression of the intermediate filament proteins vimentin and nestin. Vimentin is found in many cell types but nestin shows a more restricted pattern of expression. To improve the detection of nestin, antisera were raised against a portion of the protein expressed in bacteria and against a synthetic peptide. The antisera have been widely used to analyse cell types in the developing and adult central nervous systems of mammals. In cultured cells lacking vimentin protein, nestin was soluble, but when coexpressed with vimentin it was incorporated into intermediate filaments. The molecular basis of nestin assembly was explored by introducing the nestin gene with specific mutations into cultured cells and into transgenic mice. As with other intermediate filament proteins, deletions in conserved regions of the -helical rod domain of nestin perturbed its assembly. Deletion of the amino-terminal portion of the rod rendered the mutant subunits incapable of assembly with vimentin. A small deletion from the carboxy-terminal end of the rod affected intermediate filament morphology in the cell periphery in vimentin positive, nestin-negative cells. The same deletion reduced the ability of the mutant protein to incorporate into filaments in nestin positive cells. In transgenic animals, wild-type nestin incorporated into the endogenous vimentin-nestin filament network, but the small carboxy-terminal rod deletion remained localized near the pial endfoot in spinal cord radial glia. The two mutant proteins demonstrate that nestin interacts with other interediate filament subunits via the conserved regions of the rod domain. The results of the carboxyl-terminal rod deletion suggest that nestin may be involved in interactions between intermediate filaments and components of the subcortical cytoskeleton or the plasma membrane. 2 3 Table of Contents Page Chapter 1: Introduction 6 Figure 1. Schematic Diagram of Intermediate 36 Filament Protein Domains Figure 2. Diagram of IF consensus sites 38 Insert 1. "Multipotential Stem Cells in the 39 Vertebrate CNS" Seminars in Cell Biology, 3, pp 401-411 Chapter 2: Antibodies to Nestin 50 Figure 3 Map of Rat 401 and nestin antibody epitopes 60 Figure 4. Bacterial extract reacts with Rat 401 62 Figure 5. Western Blots comparing Rat 401 and anti-nestin 64 antisera Figure 6. Lysates of nestin-expressing mammalian cells 66 Insert 2. "Nestin expression in Embryonic Human 67 Neuroepithelium and in human Neuroepithelial Tumor Cells" Laboratory Investigation 66, pp 303-313 Chapter 3: Induction of Nestin in Embryonal Carcinoma Cells 7 8 Figure 7. Northern blot for nestin in mouse development 85 Figure 8. Western blot for nestin in P19 cells 87 Figure 9. P19 cells at 2 days of differentiation. Nestin and NFM 89 Figure 10. P19 cells at 4 days of differentiation. Nestin and NFM 91 Figure 11. P19 cells at 4 days of differentiation. Nestin and NFL 93 Figure 12. P19 cells at 4 days of differentiation. Nestin and 95 Tubulin III Figure 13. P19 cells at 4 days of differentiation. Nestin and MAP2 97 Chapter 4: Nestin Deletions 9 8 Figure 14. Schematic diagram of constructs 120 Figure 15. SW13 vimentin negative cells. Wild type nestin 122 Figure 16. SW13 vimentin negative cells. Wild type nestin 124 Figure 17. SWv-CNg21 line expressing nestin 126 Figure 18. SWv-CNg21 line expressing nestin. Later culture 128 Figure 19. Vimentin positive cells 96 hours after transfection 130 Figure 20. Vimentin positive cells expressing an 132 excess of nestin Figure 21. Stably selected nestin positive line SWv+CNg2 134 Figure 22 SWv+CNg2. Vimentin is more finely divided in nestin 136 expressing cells Figure 23. SWv+CNgl line shows marked aggregation of nestin 138 and vimentin Figure 24. SWv+CNgl line. Only cells expressing nestin have 140 aggregates Figure 25. Vimentin positive cells transfected with CNgM5 142 Figure 26. Vimentin positive cells transfected with CNgM5 144 Figure 27. Vimentin positive cell expressing high level of nestin 146 4 Figure 28. SWv+CNgM5.12, a stable line expressing mutant nestin 148 Figure 29. SWv+CNgM5.12, at lower magnification 150 Figure 30. SWv+CNgM5.13 does not show fragmentation 152 Figure 31. SWv+CNgM5.13 cells at high power 154 Figure 32. SWv+CNgM5.13 at later passage, shows retraction of 156 vimentin Figure 33. SW13 cells double stained for nestin and other 158 cytoskeletal proteins Figure 34.. Nestin promoter constructs in SW13 express strongly 160 Figure 35. Nestin amino-terminal deletion construct in SW13 162 Figure 36. NSELacZHipP2-21 cells transfected with nestin construct 164 Figure 37. Western Blot of nestin and vimentin in SW13 cells 166 Figure 38. Triton X-100 fractionation of cells 168 Chapter 5: Transgenic Animals 169 Table 1 Nestin Transgenic animals 181 Figure 39. Control for double staining 183 Figure 40. Wild-type nestin transgenic animals E.5 spinal cord 185 Figure 41. Mutant nestin transgenic embryos E.5 spinal cord 187 Figure 42 Mutant nestin animals sired by founder animal 189 Chapter 6: Conclusion 190 Biography: Martha Marvin was born in San Francisco in 1963 and recieved a Bachelor's Degree in Chemistry from the University of California, Berkeley in 1984. She worked as a chemist at Syntex Corporation in Palo Alto, CA, and on halobacterial ion channels in the lab of Dr. Roberto Bogomolni at University of California, San Francisco, from 1984-1987. Dedication IN MEMORY of Jonas Dahlstrand 1963-1994 Jonas was a treasured colleague and friend. He coaxed me out of the lab and showed me Stockholm, and his friendship made my stay there a great pleasure. His contributions to nestin research in general and to Chapter 5 in particular were invaluable. I will never forget his warped sense of humor, our conversations about literature, art and politics, and his wise advice on the health of a flying kitten. His conversations on matters of science were always illuminating. Heartfelt thanks to Urban Lendahl, who taught me molecular biology, and invited me to his lab to learn to make transgenics anyway. His collaboration made much of this work possible. Thanks of course to Ron McKay, who has made his labs productive and congenial places to learn. Thanks to Erik Nilsson for his patient teaching. May your (mouse) eggs always be sunny side up. Timothy Hayes taught me all the molecular biology I didn't learn from Urban, gave consistently wise counsel, and brought computers back from the dead. Lyle Zimmerman initiated me into the mysteries of intermediate filaments and provided comic relief. To Carolyn Smith, a thousand thanks for help with the confocal microscope. Thanks to Jim Pickel, for help with figures. The thoughtful readings of this work by my colleagues Mickey Duguich-Djordjevic, David Panchision, Tim Hayes, and Richard Josephson helped enormously in making order out of chaos. Thanks to Ed Gollin, for everything, especially January 14, 1995. This work could not have been carried out without the advice and help of the following: Bettina Winckler, Tim Hayes, Lyle Zimmerman, Richard Josephson, Jerry Yin, Mary Herndon, Chris Stipp, Hemai Parthasarthy, Oliver Brustle, Shigeo Okabe, and Erik Nilsson. 5 Chapter 1: Introduction to the Neuroepithelial Stem Cell The extraordinary complexity of the adult vertebrate central nervous system (CNS) is reached through the progressive narrowing of the developmental choices presented to the stem cells of the early neuroepithelium. In the first step a region of ectoderm is induced to commit to a neuroepithelial fate by signals from the underlying mesoderm during gastrulation. The presumptive neuroepithelium folds in on itself to generate the neural tube. Through a variety of mechanisms still not well understood, the neural tube develops rostral-caudal and dorsal-ventral axes of polarity, but for a time after the closure of the neural tube, the cells in the columnar epithelium appear as yet undifferentiated. Many lines of evidence demonstrate the existence in the early CNS of a multipotential stem cell capable of giving rise to all the cell types of the brain, but isolating and characterizing a CNS stem cell is a challenging experimental goal. In the search for developmental intermediates, markers for CNS stem cells and their differentiated progeny were required. Hybridoma technology made possible the development of specific reagents to identify cells by their gene expression. The monoclonal antibody Rat 401 was isolated in a screen for antibodies raised against E15 rat spinal cord (Hockfield and McKay, 1985). At this stage of development, some motor neurons have already terminally differentiated, but many neuronal stem cells are still proliferating. The corresponding antigen was later cloned and demonstrated to be an intermediate filament protein, now called nestin (Lendahl et al., 1990a). This antibody stained radial glia in the E15 spinal cord, but its expression began long before the birthdate of neurons, shortly after neural tube closure. Nestin first appears at E9.5 in the spinal cord and is expressed in 98% of the spinal cord cells by El 1.