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Bio 402/502 Section II, Lecture 1 The Cell Nucleus and Its Organization Dr. Michael C. Yu.

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Presentation on theme: "Bio 402/502 Section II, Lecture 1 The Cell Nucleus and Its Organization Dr. Michael C. Yu."— Presentation transcript:

1 Bio 402/502 Section II, Lecture 1 The Cell Nucleus and Its Organization Dr. Michael C. Yu

2 Outline of lectures by Dr. Yu (Section II) Lecture 1: The Cell Nucleus: an overview Lecture 2: Nuclear Processes: DNA replication/transcription Lecture 3: Nuclear Processes: Transcription/mRNA splicing Lecture 4: Nuclear Processes: mRNA processing and export Lecture 5: Nuclear protein transport Lecture 6: Chromosome Territory & Nuclear organization Lecture 7: Systems biology of the nucleus Exam Q’s: materials from assigned primary articles, lectures, and some textbook readings (available for copying in the Biological Sciences Dept Office) Exam Format: open book & open notes/journal articles

3 Nuclear functions: what are they? Diagram of a Cell’s nucleus (Website of Dr. D. Spector, CSHL) DNA replication, gene expression, etc Associated with heterochromatin Splicing factor localization ?

4 Nuclear Functions Revealed Nuclear envelope: provides the compartmentalization and structure Nucleolus: site of snRNA and ribosomal RNA maturation Heterochromatin: role in gene expression Chromosomal territory (CT): higher order organization THE MOST IMPORTANT FUNCTION: GENE EXPRESSION

5 Nuclear lamina (Website of Dr. D. Spector, CSHL)

6 Electron micrograph of nuclear membrane reveals its function Nuclear membrane: Compartmentalize the nucleus Constitutes inner membrane (IM) and outer membrane (OM) (UTMB Cell Biology) OM: contiguous with rough endoplasmic reticulum (ER)-protein synthesis. IM: proteins such as lamins are anchored to the IM.

7 More on nuclear membrane/nuclear envelope Nuclear membrane: Studded with nuclear pores (the Cell website) (Voelt et al, 2002) Nuclear lamins are building blocks of nuclear architecture.

8 (Alberts et al) Intermediate filament proteins Form meshwork at inside of inner nuclear membrane (INM), some extend into nucleoplasm Nuclear strength and architecture DNA replication and mRNA transcription Involved in apoptosis Functions of lamins (slide from Jess Hurt, HMS)

9 -Lamins are only found in nuclei of multicellular eukaryotes OrganismLamins Yeast- WormsLMN-1 FlyLamin C DmO HumansLamin B1 Lamin B2 Lamin B3 Lamin A Lamin A  10 Lamin C Lamin C2 (Stewart, Curr. Op. Gen. and Dev. 2003) Different types of lamin and organisms that have them (slide from Jess Hurt, HMS)

10 Human diseases due to mutations in the nuclear envelope (Broers, J. L. V. et al. Physiol. Rev. 86: )

11 Patients suffering from lamin disorder (progeria) Hutchinson-Gilford Progeria syndrome: accelerated aging

12 Progeria Mandibuloacral dysplasia Normal (Novelli C., TRENDS in Mol. Med. 2003) Differences in the nuclei from lamin-caused disorder Immunostaining: use of a protein-specific antibody followed by fluorescent- dye conjugated secondary antibody to detect the protein-specific antibody bound on a slide/tissue section

13 Experimental Evidence Supporting Lamin Functions Nuclear envelope assembly: Genetic studies using Drosophila, C. elegans, mouse (KO of lamin gene) Nuclear structure defects due to lamin WT Mutant } (Guillemin et al 2001) Change in the nuclear membrane architecture

14 Electron microscopy of wt and Lmna -/- MEF’s (T. Sullivan et al. J. Cell Biol. 147 (1999) ) Discontinuities in association of chromatin with nuclear envelope Continuous association of chromatin with nuclear envelope (slide from Jess Hurt, HMS) Phenotype of LMNA -/- mouse

15 Lamin provides anchorage sites for chromatin

16 Experimental Evidence Supporting Lamin Functions Genetic studies using Drosophila, C. elegans, mouse (KO of lamin gene) Nuclear structure defects due to lamin WT Mutant } (Guillemin et al 2001) DAPI (DNA) stain - sees abnormal DNA organization Provide anchorage sites for chromatin:

17 How are tissue-specific effects achieved by mutations found in all cells? Hypothesis 1: Structural hypothesis Mutations in lamins predispose all cells to fragility. Muscle cells are affected most. Falls short in lipodystrophies. Hypothesis 2: Gene expression hypothesis Disease phenotype due to alterations in gene expression that affect particular cell types. Laminopathies & Mechanism What experiments can one perform to test these hypothesis?

18 Nuclear Pore Complex (Website of Dr. D. Spector, CSHL)

19 The nuclear pores on the membrane Type of cargo transported? How is this achieved? Purpose of nuclear pores? -allows for exchange of macromolecules -NPCs are dynamic -proteins, ribosomes, RNPs, and RNAs -Via Nups (proteins of the NPC) -assembly/disassembly of cargos via exchange of GDP for GTP by Ran

20 EM of nuclear pore complex

21 The nuclear pore complex: gateway to the nucleus Bi-directional transport of macromolecules

22 Cytoplasmicfilament Cytoplasm Nucleus Cytoplasmicring Inner ring Basket Distal ring The Nuclear Pore Complex Ribosome ~150Å ~2000Å

23 mRNA mRNA RibosomalSubunits RibosomalProteins Nucleo-Cytoplasmic Transport

24 The nuclear pore complex: gateway to the nucleus All macromolecule transport are energy-dependent Non-static Will be discussed in detail in lecture #5 Gene Gating hypothesis? Functional connectivity with NPC

25 Nucleolus (Website of Dr. D. Spector, CSHL)

26 Nucleolus: a sub-organelle of the nucleus Function: site of ribosome production, rRNA processing and synthesis Not membrane-bound

27 Nucleolus: a sub-organelle of the nucleus HeLa Cells’ nucleolus Isolated nucleoli ID nucleolar proteins by mass spec (approx. 700 proteins) (Lam et al, 2005)

28 Chromosome Territories (Website of Dr. D. Spector, CSHL)

29 Chromosome Territories Individual chromosomes are organized into chromosome territories (CTs) Purpose: to facilitate/regulate gene expression Chromatins are dynamic - interactions with nuclear architecture Correlation between CT structure and function (active vs. inactive X chromosome)

30 Correlation between chromosome territories & gene activity (Verschure et al, 1999) Distribution of transcription sites in relations to CTs

31 Colocalization of genes in the nucleus for expression or coregulation (Fraser & Bickmore, 2007) Correlation between chromosome location and gene expression Cis and trans co-association Cis-interaction/trans interaction Speckle Chromatin loop Transcription factory

32 Outstanding questions on chromosome territories How do chromosomes find their place in the nucleus? How do chromosomes form this higher-order structure? Will be discussed in detail in lecture #6 Mechanism of chromosome positioning? Thoughts on how chromosome positioning affects gene expression?


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