Lesson 1-INTRODUCTION TO CELL BIOLOGY 2023-2024 PDF

CELL BIOLOGY AND HUMAN GENETICS Ve más allá Academic Year 2023-2024 DAVID BALLESTEROS PLAZA Department of Pre-clinical Dentistry (Building A) E-mail address: davidalberto.ballesteros @universidadeuropea.es Ve más allá Academic Year 2023-2024 Cell Biology Lesson 1 INTRODUCTION TO CELL BIOLOGY 1. 2. 3...

CELL BIOLOGY AND HUMAN GENETICS Ve más allá Academic Year 2023-2024 DAVID BALLESTEROS PLAZA Department of Pre-clinical Dentistry (Building A) E-mail address: davidalberto.ballesteros @universidadeuropea.es Ve más allá Academic Year 2023-2024 Cell Biology Lesson 1 INTRODUCTION TO CELL BIOLOGY 1. 2. 3. 4. 5. 6. Definition of Cell Biology and Historical perspective of cell Biology. Cell theory Basic properties of the cell Structure and organization of cells Acellular forms: viruses, viroids and prions Tools to study cells: Optical and Fluorescence Microscopy. Electronic Microscopy. Stem cells. Cell cultures. 1. Historical Perspective of Cellular Biology : The Cell Theory. Cell Biology : Historical Background BIOLOGY is the scientific discipline that studies the structure, physiology, evolution, distribution and relations of living organisms. CELL BIOLOGY is the área of biology that studies the structure and function of cells, assuming that the cell is the fundamental unit of life. HISTORICAL ORIGIN : The development of microscopy tools and techniques allowed the detailed observation of the first structures in nature and the first cells (XVIII century). Cell biology emerged in parallel to other sciences like Citology and Histology and thanks to advances in Medicine and Physiology. As microscopy techniques improved, so did our knowledge of the microscopic structure of simple organisms and/or tissues. This gave rise to the Cell Theory. The Cell Theory was formulated by Schwann and Schleiden Theodor Schwann (1810-1882) He was a German zoologist and came to the conclusion that the cell is the fundamental unit of the structure and organization of all living organisms. Two new disciplines emerged : Cytology, to study cells and Histology to study the structure of tissues. He also discovered the myelin sheath (Schwann´s sheath) in the peripheral nerve fibers. Matthias Jakob Schleiden (1804 -1881) He was a German botanist that shared Schwann´s views on the relevance of cells. A year after Schleiden published a cellular theory on plants, Schwann expanded his theory to include animals, thus unifying Botany and Zoology under a common theory. Rudolf Virchow (1821-1902) Medical doctor and pathologist, that established the omnis cellula ex cellula theory (all cells come from pre-existing cells). He also discovered that the starting point of a disease is the cell and not the organ. He is considered the father of Cellular Pathology.  He established the cell division as the key event in organismal reproduction or proliferation.  He was also the first to show that the Cell Theory applied no only to healthy tissues but also to pathological tissues. He was a pioneer of the modern concept of pathological process.  Louis Pasteur (1822-1895) One hundred years after the discovery of microorganisms by Leeuwenhoek, the spontaneous generation theory was still valid. Spontaneous generation is the hypothesis that some vital force contained in or given to organic matter can create living organisms from inanimate objects. Louis Pasteur ended the debate of the spontaneous generation with his famous swan-neck flask experiment, which allowed air to contact the broth. Microbes present in the dust were not able to navigate the tortuous bends in the neck of the flask. Pasteur proved to the world that life could only come from other life. Microorganisms only appeared in those flasks that had the broken neck. Demonstration of the Cell Theory Louis Pasteur Swan-neck bottles 1860 The Cell Theory was initially proposed by Shleiden and Schwann and later completed by Virchow. 1. All living creatures are formed by cells. Cells are therefore THE STRUCTURAL UNITS of organisms. 2. All cells come from preexisting cells, through the process of cell division (Omnis cellula ex cellula). Therefore cells are the REPRODUCTIVE UNIT. 3. A cell sustain accomplish all the vital functions. Therefore the cell is also the PHYSIOLOGICAL UNIT of a living organisms. 4. Each cell contains all the hereditary information required to control its own life cycle, development and functions and to transfer that information to the next cell generation. Therefore a cell can also be considered the basic GENETIC UNIT. 2. Basic Properties of the Cell Basic Properties and Functions of Cells Production and use of energy Nutrition Growth and evolution Transport Reproduction Organization Response to stimulus Comunication Differentiation Self-regulation capacity  Cells are complex and highly organized. Different cells perform different functions  Cells have metabolic capacity : they produce and use matter and energy.  Cells can self-replicate and proliferate. Cells have a genetic program that allows them to replicate and transfer the genetic information to new cells.  Cells can relate and communicate with the environment and with other cells: they exchange matter, energy and information with extracellular medium and other cells.  Cells are capable of responding to different stimuli: they can change their shape and move.  Cells can autoregulate : they divide only under optimal environmental conditions or they can undergo programmed cell death if they are severely damaged. 3. Structure and Organization of Cells Cell types. Similarity and Diversity Blood cells Neurons Plant cells Eukaryotes and Prokaryotes Bacteria Prokaryotes Are the Most Diverse and Numerous Cells on Earth  Prokaryotes are typically spherical, rodlike, or corkscrew-shaped  They are also small—generally just a few micrometers long  Prokaryotes often have a tough protective coat, or cell wall, surrounding the plasma membrane, which encloses a single compartment containing the cytoplasm and the DNA.  Quick proliferation.  Populations of prokaryotic cells can evolve fast, rapidly acquiring the ability to use a new food source or to resist being killed by a new antibiotic. PROKARYOTIC CELL Capsule or glycocalix - outer sticky protective layer attached to the cell wall. Cell Wall - rigid structure that helps the bacterium maintain its shape. It is different from the wall of a plant cell. Plasma membrane - separates the cell from the environment. Mesosome membrane. - infolding of plasma Nucleoid - region where DNA is found. Bacterial chromosomal DNA is circular and double-stranded. Bacteria may also contain small non-chromosomal plasmid DNA (resistance to antibiotics) Cytoplasm - semi-fluid cell interior containing metabolic enzymes and ribosomes for protein synthesis BACTERIAL FLAGELLUM : to propell the cell through its surroundings. A single bacterium can have one or more. The structure, composition and movement is different from the flagellum of eukaryotic cells. The main component of bacterial flagellum is the protein Flagellin. FIMBRIA: filamentous structure, shorter tan flagellum and used for adhesion to other cells or to a surface. PILI: extension that bacteria use to exchange genetic material (plasmids) with other bacteria. Vibrio cholerae THE BACTERIAL GLYCOCALIX OR CAPSULE: External sugar-rich coating attached to the cell wall. Provides resistance to adverse environmental factors. It may also restrain phagocytosis and binding of host antibodies. The capsule facilitates bacterial adherence to dental surfaces and improves adhesion to other bacterial cells. Therefore, the capsule plays a key role in formation of the dental cariogenic plaque. Not all bacterial strains have a capsule. EUKARYOTIC CELL STRUCTURAL ORGANIZATION OF EUKARYOTIC CELLS The NUCLEUS holds and stores the cell´s genetic and heritable information. The CYTOSOL is an aqueous gel full of molecules. The ENDOMEMBRANE SYSTEM generates specialized compartments with different functions : Endoplasmic Reticulum, Golgi Apparatus, Lysosomes, Peroxisomes, Vesicles. MITOCHONDRIA produce energy for the cell from nutrients. The CYTOSKELETON provides a flexible scaffold to maintain the cell shape, anchor organelles, facilitate intracellular transport, and support cell movements. Compartmentalization PROKARYOTIC CELLS EUKARYOTIC CELLS LACK NUCLEAR ENVELOPE WITH NUCLEAR ENVELOPE SINGLE CHROMOSOME MULTIPLE CHROMOSOMES LACK NUCLEOLUS WITH NUCLEOLUS AMITOTIC (binary) DIVISION DIVISION : MITOSIS / MEIOSIS LACK MITOCHONDRIA WITH MITOCHONDRIA LACK CHLOROPLASTS CHLOROPLASTS PRESENT IN PLANTS LACK MEMBRANE ORGANELLES WITH MEMBRANE ORGANELLES Features from Eukaryotes shared with Prokaryotic cells Rigid cell wall : Plant cells, some Fungi, some Protists Animal cells lack cell wall Plasma membrane Cytoplasm with ribosomes Genetic material PROKARYOTES EUKARYOTES Organisms Bacteria, cyanobacteria Protists, Fungi, plants, animals Size 1-10 µm 10-100 µm Metabolism Aerobic, anaerobic Aerobic Respiratory Chain Location Mesosomes Mitochondria Endomembrane System NO, unique space Yes, compartmentalization Cholesterol NO YES Cell Wall Yes (peptidoglycan) Only in plant cells (cellulose) Ribosomes 70S 80S DNA Circular Lineal, bound to histones Cell Division Non mitotic (binary fission) DNA replication and condensation. Mitotic. Motion Flagellum (Flagellin) Several mechanisms (cytoskeletal Tubulin) 4. Acellular Forms: Viruses,Viroids, Prions ACELLULAR FORMS: VIRUSES From the latin word virus, toxin or poison  Viruses are obligatory intracellular parasites  Viruses infect host cells, produce multiple copies of themselves, and destroy the cell.  Viruses are very small with a size of 24-300 nm (electron microscope).  They usually have geometric shapes. Many have been crystallized. DNA Capsid Proteins  Genetically modified viruses are often used in cell biology research to carry foreign DNA into a cell of interest. For the same reason viruses have potential as therapeutic agents. https://www.khanacademy.org/science/biology/biology-of-viruses/virus-biology/v/viruses VIRUS STRUCTURE Nucleic acid, containing the genetic information of the virus for selfreplication. Types of nucleci acid: -DNA, -RNA Capsid (are proteins) which involves the nucleic acid. The capside is organized in capsomers. These capsomers can be organized to give different type of structures. Several virus contain an outer membrane, that is a lipid bilayer (belongs to the host cell). Naked virus: without outer membrane Enveloped virus: the outer membrane is needed to infect the cells. 3 TYPES OF VIRUS: 1- Bacteriophage 2- animal or plants virus 3- retrovirus RNA Viruses that infect prokaryotes CLASSIFICATION animal viruses From genomic nucleic acids (virus genomes) To Translation into viral proteins using host machinery (viruses use different strategies) Classification based on viral genomes. Great variety of morphologies and genomes (dsRNA (retroviruses), ssRNA, dsDNA, ssDNA) SARS-Cov-2 structure Coronavirus organization. A model of the coronavirus structure showing the organization of the spike (S), membrane (M), and envelope (E) glycoproteins. The RNA is protected by a helical capsid of N protein monomers. CREDIT: KATHARINE SUTLIFF/SCIENCE Icosaedric viruses Helicoidal viruses Complex viruses adenovirus bacteriophage HIV Virus de la gripe Poliovirus Virus del herpes simple Coronavirus Papillomavirus Ebola ACELLULAR FORMS: VIROIDS Viroids are the smallest known pathogen (1/10 of virus size). Viroids are essentially naked, circular, singlestranded RNA molecules that do not encode proteins. Viroids don´t have a capsid. Viroids replicate their RNA using the host cell transcription machinery. Viroids only infect plants; some cause economically important diseases of crop plants, while others appear to be benign. Example: cadang-cadang The coconut tree disease Cadang-Cadang is caused by a viroid. They have been proposed to be relics of the “RNA World”. ACELLULAR FORMS: PRIONS Prions are the only known infectious agents without nucleic acids : prions are infectious proteins. They cause several neurodegenerative diseases, mostly Spongiform Encephalopaties (Creutzfeldt-Jakob Disease and Kuru in humans, Scrapie in sheep, Bovine Spongiform Encephalopathy or Mad Cow disease ) CAUSE OF THE DISEASE : The infectious protein (called PrPSC) causes the misfolding of the normal cellular prion protein (PrPC), a glycoprotein expressed mainly in nervous cells. The misfolded protein becomes infectious and can cause misfolding of other normal prion protein molecules. In addition, misfolding leads to aggregation and eventually formation of plaques in the brain tissue. PrPC PrPSC Brain cortex sample of a CJ patient CONSEQUENCES OF PRION DISEASES: The abnormal folding of the prion proteins leads to brain damage. Prion diseases are progressive and always fatal. https://www.youtube.com/watch?v=Xws0_I-xyOI 5. Cell Biology Tools and Techniques :Microscopy & Cell culture. Microscopy Resolution Limit of human eye : 100 μm (0,1mm) 1m 1 mm Eye and simple lenses 10 µm Resolution Limit of optical microscope : 0,2 μm 1 µm Optical microscope 100 nm Resolution Limit of electron microscope : 1 nm 10 nm Electron microscope Tissues Cell components Cells Viruses Bacteria 1 nm Optical or Light Microscopy Magnification : up to 1000 x Resolution : approx. 0,2 μm TRANSMITTED LIGHT MICROSCOPY Ocular Phase Contrast Bright Field Live cells or fixed tissue sections BF Differential Interference Contrast Live cells with enhanced contrast PhC Objective Specimen Light Beam DIC Condenser Light Source Optical or Light Microscopy REFLECTED LIGHT MICROSCOPY Fluorescence Microscopy Fixed Cells/tissues or Live Cells expressing fluorescent proteins Confocal Microscopy Emission Filter For sharper images of thicker samples Excitation Filter Sample Excitation at a specific wavelenght and emission at a different wavelenght Require the use of Fluorescent Dyes that absorb light at one wavelength and emit it at another, longer wavelength. IMMUNOCYTOCHEMISTRY Common technique based on the use of specific antibodies to detect the presence and location of a particular molecule in a cell or tissue. These antibodies can be covalently bound to a fluorescent probe or to an enzyme.  Enzymatic Immunodetection antibody is bound to an : the enzyme (peroxidase, alkaline phosphatase) that transforms its substrate into a coloured product (chromogen). Detection can be direct (primary antibody bound to enzyme) or indirect (secondary antibody bound to enzyme).  Fluorescence Immunodetection (immunofluorescence) : the antibody is bound to a fluorescent probe (fluorochrome). Detection can be direct or indirect. Direct IF Indirect IF Secondary Antibody Primary Antibody Cell /Tissue Cell /Tissue Primary Antibody detects the molecule of interest Secondary Antibody detects the primary antibody (anti-rabbit IgG, anti-mouse IgG … Enzymatic Immunodetection Fixed Tissue Sections (immunohistochemistry) Immunofluorescence Fixed Cells or Tissues Live cells for surface antigens Expression of Fluorescent Proteins (proteins tagged with a fluorescent protein) Bright-field microscope Fluorescence microscope. blue (ECFP) yellow (EYFP) Different stages of a mouse embryo Electron Microscopy Magnification : up to 100000 x Resolution : approx. 1-2 nm  Transmission Electron Microscopy (TEM) Uses a beam of electrons instead of a beam of light Electrons pass through the specimen The sample must be very thin Electron Microscopy  Scanning Electron Microscopy (SEM) Electrons do not pass through the specimen. The specimen is bombarded with a beam of electrons and electrons are scattered or emitted from the surface of the specimen and collected to generate a threedimensional image. Electron Microscopy CELL CULTURE Growth of cells or cellular models under CONTROLLED CONDITIONS.  Cell culture is performed on artificial media prepared by mixing purified components or complex organic solutions. EXAMPLE OF CULTURE MEDIUM : DMEM Bicarbonate buffer,  Maintenance of pH Salts, pyruvate, glucose, aas, vitamins…  Nutrients Fetal Bovine Serum (5-15%) Growth factors- Proliferation Antibiotics/Antimicotics (10%)  sterility Phenol red, to detect medium acidification  Cell culture requires specific equipment (cell incubator) to maintain the appropriate physico-chemical conditions : temperature, humidity, CO2 and O2.  Most animal cells derived from solid tissues are ADHERENT and require a solid support to grow. This support is provided by the surface of a plastic tissueculture dish.  Other cells (blood cells for example) can grow IN SUSPENSION Types of Cell Culture PRIMARY CULTURES  Derived from healthy tissue  Cells maintain their normal morphology and functionality  They show contact inhibition.  Normal number of chromosomes (2n) : diploid karyotype.  Limited proliferation : after a certain number of cell divisions, cells stop growing. Primary hepatocytes (Rat liver) CELL LINES  Derived from tumors or obtained through transformation of a primary culture (transfection with oncogenes or carcinogenic treatment).  They do not show contact inhibition  Aberrant number of chromosomes :  Unlimited proliferation : they can undergo limitless cell divisions and are therefore considered “immortal cultures”. HepG2 line (Human hepatocarcinoma) 6. STEM CELLS http://stemcells.nih.gov/info/basics STEM CELLS Stem cells are undifferentiated cells that can turn into a wide variety of specialized cells Stem Cell  Self-renewal : they can divide to produce new copies of themselves. Self-Renewal All stem cells share three basic characteristics:  Capacity to differentiate into other cell types (at least in two …)  Capacity to colonize and originate new tissues… Stem cells can repair damaged tissues or replace differentiated cells that cannot divide but need to be renewed (skin cells, blood cells …). Differentiated Cells STEM CELL DIVISION Stem cells can undergo SYMMETRIC or ASYMMETRIC divisions.  Symmetric Division : A stem cell produces two stem cells or two differentiated progenitor cells. Useful to increase the stem cell population or the population of progenitors in case of injury.  Asymmetric Division : A stem cell gives rise to one stem cell and one differentiated progenitor cell. Useful for normal homeostasis of tissues. Stem cells Differentiated Progenitors CLASSIFICATION OF STEM CELLS Stem cells can be classified according to their differentiation potential  Totipotent Stem Cells: cells with the potential to create a whole organism. All the cells from the very early stages of embryo development (3-4 days after fertilization) are totipotent.  Pluripotent Stem Cells: cells with the potential to produce cells of the three germ layers: ectoderm, mesoderm and endoderm. They are found in the inner mass of the blastocyst (pre-implantation stage embryo)  Multipotent Stem Cells: can give rise to several types of cells of a particular lineage. Examples of multipotent stem cells include those in the brain that give rise to different neural cells and glia or haematopoietic stem cells, which can give rise to all different blood cell types, but they cannot create brain cells.  Unipotent Stem Cells: an unipotent stem cell will differentiate into only one specific cell. The tissue-specific cell will then provide functional and structural components to a body tissue or organ. Stem Cells Types Based on their Origin 1 Embryonic stem cells (ES) 2 Non Embryonic and Non Adult stem cells : Stem Cells from Umbilical Cord (UCS) or Amniotic Fluid (AFS) 3 Adult stem cells Haematopoietic stem cells (HSC) Mesenchymal stem cells (MSCs) Tooth stem cells (TS) Adipose stem cells … 4 Induced Pluripotent stem cells (iPS). Differentiated adult cells genetically reprogrammed into pluripotent cells. EMBRYONIC STEM CELLS: Primitive (undifferentiated) cells obtained from the inner mass of preimplantation- stage embryos (blastocyst). They can be obtained from unused, embryos produced by “leftover” in vitro fertilization, and can develop into cells and tissues of the three primary germ layers : they are PLURIPOTENT. Ethical issues on the use of embryos has precluded their widespread study, especially in humans. ADULT STEM CELLS : they are among stem cell found differentiated cells in a tissue or organ. An adult stem cell can selfrenew and can differentiate to yield some or all of the major specialized cell types of the tissue or organ. However they have a limited capacity for both self renewal (in the laboratory) and differentiation. The primary role of adult stem cells in a living organism is to maintain and repair the tissue where they are found. Induced Pluripotent Stem Cells (iPS) They are adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by artificially expressing factors important for maintaining the defining properties of embryonic stem cells. Like ES cells, iPS cells are PLURIPOTENT. The four required reprogramming factors are : Oct4, Sox2, cMyc and Klf4. They are transcription factors that control the expression of other genes. Takahashi, K. and Yamanaka, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663–676. Dr. Yamanaka receive the Nobel Prize Award in 2012 for this discovery. Tooth Stem Cells (TS) These stem cells can be found in the dental pulp, periodontal ligament or other dental structures They are MESENCHYMAL STEM CELLS and as such have the potential differentiate into many different cell types ; chondrocytes, osteoblasts, adipocytes, myocytes, neuronal cells, cardiomyocytes … These stem cells are MULTIPOTENT. Use of Tooth Stem Cells in Odontology  Tooth stem cells could be used to regenerate dental structures.  The mayor advantage is that TS are AUTOLOGOUS CELLS, that is, cells obtained from the patient. Therefore, there is no risk for immune rejection.  In addition, they are fairly easy to obtain through non invasive methods. TS adipocytes odontoblasts neurons ESSENTIAL ORIGIN S XIX: Schleiden and SchwannCELLULAR THEORY; Virchow  division; Pasteur ruled out spontaneous generation (gooseneck flask sterility experiments) CELLULAR THEORY: the cell is the structural, physiological, reproductive and genetic unit of the living being. CELL PROPERTIES: Complex and organized, have metabolism, can be self-perpetuate, relate to the environment and other cells, can react to stimuli, can self-regulate GENERAL STRUCTURE OF THE PROKARYOTE: 1 μm, types of walls, mesosomes, nucleoid, fimbriae, flagella, pili, GLUCOCALIX (role in the acquired film). GENERAL STRUCTURE OF EUKARYOTES: 10-100 μm, organelles. Differences with prokaryotes ACELLULAR FORMS: Viruses: