Undergraduate – Graduate Nucleus and Mitosis Histology Lecture Series VIBS 443 and VIBS 602 Larry Johnson, Professor Veterinary Integrative Biosciences Texas A&M University College Station, TX 77843 Objectives Characterize the interphase nucleus including the cell cycle, nucleolus, chromatin, nuclear envelope, and nucleocytoplasmic interactions. Characterize mitosis including structures with definitions, mechanisms of chromosome movements, and cytokinesis. Remember the proposed evolutionary involvement of the cell membrane in organelle formation. Eukaryotic cell Eukaryotic cell Nucleus ( interphase nucleus) Microtubules Intermediate filaments Stabilize nuclear envelope Nucleus - archive of the cell Archive of the cell - nucleus encodes for all proteins and secretory products. All somatic cells have the same codes regardless of their final function in the body (e.g., nerve, epithelium, CT, muscle). Heterochromatin - Nucleus Cells in an individual are the same in their condensed genome, but they differ in its expression. chromatin Euchromatin – dispersed for active transcription Nucleus Source of major informational macromolecules that direct protein synthesis. tRNA, mRNA, and, rRNA Nucleus Chromatin – Nucleoprotein - stain basic dye Nuclei Cell cycle Generation time varies with cell type – 4-6 days in intestine absorptive cells – Year in nerve cells Cell cycle GAP 2 G1/S/G2/M M – Mitosis GAP 1 S – DNA synthesis GAP 1 GAP 2 Cell cycle Shown by differential Morphology varies with interference contrast the cell cycle. microscopy. Dead stained cells Bright field Live unstained cells Phase contrast Nomarski differential interference contrast Dark field Phase contrast light microscopy Cell cycle The cell cycle - cells round up for mitosis • http://www.youtube .com/watch?v=rgLJ rvoX_qo • http://www.youtube .com/watch?v=DD 3IQknCEdc&featur e=related • http://www.youtube.com/watch?v=Q6ucKWIIFmg&feature= related Nucleolus Cell fractionation to study nucleoli morphology - ribosome production Fibrous 50 A° 45S 18S + protein 40S (small subunit) Granular 120 to 150 A° 45S 28S + protein 60S (large subunit) Nucleolus Metabolic activity of cell reflected in size of nucleolus Pituitary cell If axon is cut, size of nucleolus increases by 50% Oocyte Sertoli cell Leydig cell Nerve cell Nucleolus To study the pathway of ribosome formation: Pulse chase experiment – 3H uridine for RNA Temporal appearance of radioactivity revealed that 3H uridine first was seen over the nucleus, then nucleolus, and finally over the cytoplasm. Nucleolus Ribonucleoproteins - Have an affinity for basic dyes and are the most conspicuous of stained nuclear organelles. Nucleolus Disperses in cell division Nucleolus Contains genes for ribosomal RNA. Nucleolus Is essential for protein synthesis destined for cytoplasm. Note contribution of: Cytoplasm Nucleus Nucleolus to ribosomal production Nucleolus Summary Pulse chase experiment – 3H uridine for RNA, Temporal appearance of radioactivity revealed that 3H uridine first was seen over the 1) Nucleus, 2) Nucleolus, and finally over the 3) Cytoplasm 3H uridine for RNA Chromatin DNA double helix w/ protein – 20 A° thick Mitosis 500-1000 A° thick Chromatin 100s of miles of DNA in single cell packaged for mitosis. Beads on a string Chromatin Heterochromatin – sperm cell nucleus, inactive and dark Euchromatin – nerve cell nucleus, active and light Chromatin Histones and other proteins – from cytoplasmic ribosomes Polyribosomes Nuclear envelope Cell membrane is involved in organelle formation. Outer membrane - continuous with RER Inner membrane space - cisterna of RER Nuclear envelope Nuclear pores – nuclear cytoplasmic exchange Pore size – 40 to 100 A° Number = 2000/nucleus Nuclear envelope Nuclear pores – Gold study 80-100 A° Nucleoplasmins are molecular chaperones that transfer the DNA to the histone-DNA complexes by binding to the histones. Nuclear envelope Nuclear pores Ribosomal large subunit 120 to 150 A° squeezes through 40 TO 100 A° Pore size = 40 to 100 A° Nuclear envelope Nuclear pores Nuclear envelope Nuclear pores DNA not associated w/ pores RNA always associated w/ pores Nuclear envelope Fibrous lamina or (nuclear lamina) Intermediate filaments Annulate lamellae = nuclear pores like structures in RER Why chromosomes, kinetochores, and centrioles? Chromosomes – super coil prevents errors during mitosis Kinetochores – most important part for division or segregation of chromosomes Why chromosomes, kinetochores, and centrioles? – Many plants have no centrioles – If a centriole is removed, the cells still divide – Real function unknown, except they are needed to be present to be pasted to the daughter cell for what ever their function is Microscopy that allows observation of live cells. Why do cells divide? Allow organism to increase in mass Replace damaged cells Reduce size of cell - improved diffusion Why do cells divide? Avoid aging – build up of waste products. If a portion of a cell is removed, it prevents cell division. Mitosis Defined - cell division in which duplicated chromosomes divide into daughter cells that have identical chromatin content Mitotic figures in gut epithelium Mitosis Cell division in gut epithelium MITOSIS Definitions – Karyokinesis - nuclear division – Cytokinesis - division of cytoplasm Completion in 30 min to 2.5 hrs Cells in mitosis in intestinal epithelium Chromosomes - rod-like organelles, visible during cell division Number of chromosomes – Diploid - 2n - full complement – Haploid - 1n - half complement – Polyploid - more than full complement (Down’s syndrome) Chromosomes – rod-like organelles, that are visible during cell division Gene - hypothetical units of inheritance – Linearly arranged along length of DNA – Gene locus – Deletion – Translocation Three main events in mitosis 1. Orientation (after chromosomal condensation and nuclear envelope breakdown) 2. Alignment 3. Segregation MITOSIS ORIENTATION SEGREGATION ALIGNMENT Events in mitosis Cytokinesis Events in mitosis Cytokinesis Events in mitosis Cytokinesis Events in mitosis Cytokinesis Events in mitosis Centrosome separation Mechanisms of chromosome movement Microtubule assembly - disassembly model Sliding microtubules model • http://www.youtube.com/watch?v=VlN7K1- 9QB0&feature=related • http://www.highlighthealth.com/did-you-know/mitosis-and-meiosis/ Chromosome movement Kinetochore, polar, and astral microtubules Chromosome movement Chromosome movement Chromosome movement Three contributions 3 1 Polar Kinetochore 2 Astral 1) polar, 2) astral, and 3) kinetochore microtubules Chromosome movement http://www.youtube.com/watch?v=2WwIKd yBN_s&feature=related http://www.youtube.com/watch?v=6dMlde9akBk&feature=related Developing ovarian follicle has many cells in mitosis. Mitosis Next time Peripheral blood Many illustrations in these VIBS Histology YouTube videos were modified from the following books and sources: Many thanks to original sources! • Bruce Alberts, et al. 1983. Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY. • Bruce Alberts, et al. 1994. Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY. • William J. Banks, 1981. Applied Veterinary Histology. Williams and Wilkins, Los Angeles, CA. • Hans Elias, et al. 1978. Histology and Human Microanatomy. John Wiley and Sons, New York, NY. • Don W. Fawcett. 1986. Bloom and Fawcett. A textbook of histology. W. B. Saunders Company, Philadelphia, PA. • Don W. Fawcett. 1994. Bloom and Fawcett. A textbook of histology. Chapman and Hall, New York, NY. • Arthur W. Ham and David H. Cormack. 1979. Histology. J. S. Lippincott Company, Philadelphia, PA. • Luis C. Junqueira, et al. 1983. Basic Histology. Lange Medical Publications, Los Altos, CA. • L. Carlos Junqueira, et al. 1995. Basic Histology. Appleton and Lange, Norwalk, CT. • L.L. Langley, et al. 1974. Dynamic Anatomy and Physiology. McGraw-Hill Book Company, New York, NY. • W.W. Tuttle and Byron A. Schottelius. 1969. Textbook of Physiology. The C. V. Mosby Company, St. Louis, MO. • Leon Weiss. 1977. Histology Cell and Tissue Biology. Elsevier Biomedical, New York, NY. • Leon Weiss and Roy O. Greep. 1977. Histology. McGraw-Hill Book Company, New York, NY. • Nature (http://www.nature.com), Vol. 414:88,2001. • A.L. Mescher 2013 Junqueira’s Basis Histology text and atlas, 13th ed. McGraw • Internet images and videos on biological presentations .