Introduction to Histology PDF

INTRODUCTION TO HISTOLOGY TODAY’S TOPICS Light microscope Cell & tissue culture (H&E) Interpretation of the structure of tissue sections INTRODUCTION Histology studies tissues, and their arrangements in organs. Tissues have 2 components: cells and ECM (Extracellular matrix) ECM support...

INTRODUCTION TO HISTOLOGY TODAY’S TOPICS Light microscope Cell & tissue culture (H&E) Interpretation of the structure of tissue sections INTRODUCTION Histology studies tissues, and their arrangements in organs. Tissues have 2 components: cells and ECM (Extracellular matrix) ECM supports the cells and consists of various macromolecules In order to study tissues, they first need to be prepared. The most common method of which is slicing them in ‘sections’ and preserving with ‘fixative’ such as paraffin for maintaining shapes of structures. However this is not always possible as this process can change the tissue. TISSUE PREPARATION Fixation preserves tissue and prevents degradation by the enzymes released from the cells and microorganisms. Done by placing samples in solutions of chemicals that cross-link proteins and inactivate degradative enzymes Dehydration is done by placing tissue in highly concentrated alcohol solution Clearing is the process of alcohol removal by solvents Infiltration is placing tissues in melted paraffin, until they get completely infiltrated by it Embedding: paraffin is allowed to harden Trimming: resulting sample is sliced to expose tissue LIGHT MICROSCOPE Antonie van Leeuwenhoek is best known for his pioneering work in microscopy. TYPES OF MICROSCOPY: BRIGHT-FIELD MICROSCOPY Bright-Field Microscopy uses external light that passes through the preparation. Includes an optical system and mechanisms to move and focus the specimen Condenser focusing light on the object to be studied Objective lens enlarging and projecting the image Resolving power, defined as the smallest distance between two structures at which they can be seen as separate Resolving power of light microscope is 0.2 μm (magnified 1000-1500x) Smaller objects like Ribosome, Cytoskeleton filament can’t be seen by it TYPES OF MICROSCOPY: FLUORESCENCE MICROSCOPY Fluorescence: certain cellular substances when irradiated by proper wavelength, emit longer wavelength. Usually (UV) Fluorescent compounds with affinity for specific cell macromolecules may be used as fluorescent stains Acridine orange, which binds both DNA and RNA TYPES OF MICROSCOPY: PHASE-CONTRAST MICROSCOPY Allow the examination of cells without fixation or staining Based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices These changes are used by the phase-contrast system to cause the structures to appear lighter or darker in relation to each other TYPES OF MICROSCOPY: CONFOCAL MICROSCOPY With a regular bright-field microscope, the beam of light is relatively large and fills the specimen. Confocal microscopy achieves high resolution and sharp focus. Uses a laser as a light source. And a computer driven aperture hole. TYPES OF MICROSCOPY: POLARIZING MICROSCOPY Structures made of highly organized subunits When normal light passes through a polarizing filter, it exits vibrating in only one direction. If a second filter is placed in the microscope above the first one, with its main axis perpendicular to the first filter, no light passes through. Tissue structures containing oriented macromolecules are located between the two polarizing filters, their repetitive structure rotates the axis of the light emerging from the polarizer ELECTRON MICROSCOPY Based on interaction of tissue components with beams of electrons. The wavelength in an electron beam is much shorter than that of light, allowing a 1000x increase in resolution. Transmission Electron Microscopy and Scanning Electron Microscopy. CELL & TISSUE CULTURE Live cells can be maintained and studied outside the body in culture (in vitro) Culture allows the observation under a phase-contrast microscope Cells and tissues are grown in complex solutions of known composition Serum or specific growth factors are added Most cells obtained from normal tissues have a finite, genetically programmed life span Transformation changes that make cell immortal (Usually oncologic) INTERPRETATION OF STRUCTURES IN TISSUE SECTIONS Certain steps in the obtaining procedure may distort the tissues slightly. Artifacts Example: shrinkage of cells or tissue regions produced by the fixative, by the ethanol, or by the heat needed for paraffin embedding. Artifacts include: artificial spaces, small wrinkles, stains. A single stain can seldom demonstrate well nuclei, mitochondria, lysosomes, basement membranes, elastic fibers, etc. Therefore multiple stains are required. Loss of 3D image. H&E STAIN Most cells as well as ECM is completely colorless and they must be stained. Hematoxylin and eosin is often gold standard for visualization. The hematoxylin stains cell nuclei a purplish blue, and eosin stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors. USED LITERATURE Junqueira’s Basic Histology Text and Atlas 2018 15th ed. Pages 1-15 BRS Cell Biology Histology 2018 8th ed. Thank You  Cell, Plasma membrane, Transmembrane proteins/transport, Membrane organelles Used literature Junqueira’s Basic Histology 15th ed. Pages 17 - 42 BRS Cell Biology Histology 8th ed. Pages 1 – 35 Do not include Cytoskeleton and Nucleus  Cells and tissues All tissues that make up organs are comprised of ECM and cells. Cells are the basic structural and functional units, the smallest living parts of the body. Animal cells are Eukaryotic, meaning they have nuclei surrounded by cytoplasm, which contains organelles and cytoskeleton. Bacterial cells however, lack nuclei and usually have cell walls around plasmalemma (plasma membrane). Cells undergo differentiation during which they express sets of genes that mediate their cytoplasmic activities and make them more efficient in specialized functions. (for example muscle cells). Plasma membrane Also called cell membrane or plasmalemma, envelops every eukaryotic cell. Contains: phospholipids, cholesterol, and proteins, with oligosaccharide (oligo - few) chains covalently linked to them. Functions as a selective barrier regulating the passage of materials into and out of the cell and facilitating the transport of specific molecules. Also keeps ion content stable. Plays a key role in cell to environment interactions, by carrying out recognition and signaling functions.  Plasma membrane: Composition Some plasma proteins integrins are linked to both cytoskeleton and ECM components to allow continuous exchange of influences, in both directions. Thickness ranges from 7.5 to 10 nm, therefore is only visible with electronic microscope Phospholipids are amphipathic (have both hydrophilic and hydrophobic parts). The fatty acids repel water, while their heads attract it. Due to this nature of phospholipids they are most stable and arranged in double layer where fatty acid tails face each other (with cholesterol packed between them) while hydrophilic heads face out towards water. Outside the double layered plasma membrane is the third layer glycocalyx, composed of glycolipids (lipids with sugar residue).  Plasma membrane: Proteins Integral proteins are incorporated directly within the membrane, while peripheral proteins are bound to one of the surfaces. Some integral proteins protrude from either side of the plasma membrane and contribute to the glycocalyx, these protrusions are referred as receptors, which allow complex interactions such as cell adhesion, cell recognition, and the response to protein hormones. Their distribution is different on either side of the membrane, making it asymmetric. Studies have shown that some proteins are not bound rigidly and can move, forming a fluid mosaic model of the membrane. While proteins that are part of larger enzyme complexes are usually less mobile and are located on specialized membrane patches termed lipid rafts. Transmembrane Proteins & Transport Plasma membrane is site where materials are exchanged between the cell and environment. Exchanges of small molecules happen via following methods: Diffusion: Small, nonpolar, lipophilic molecules directly cross the membrane readily. Channels: Multipass proteins that form pores that selectively pass ions (Na+, Ca+, K+) and small molecules. Water molecules pass through Aquaporins. Carriers: Transmembrane proteins that bind small molecules and move them via conformational changes. Above mentioned methods work down the concentration gradient, therefore are passive and require no energy. In contrast, Pumps use active transport, utilizing energy from ATP and are often referred as ATPases.  Endocytosis: moving in Macromolecules (large) usually enter and exit the cell, while enclosed by part of plasma membrane (often after binding to a specific receptor). These process forms a vesicle (bubble). Phagocytosis: “cell eating” is when cells ingest particles (macrophages/neutrophils). When a foreign particle such as bacteria gets encircled by cell plasmalemma end cytoplasm, it gets entrapped in phagosome, which later merges with lysosome, which degrades everything within it. Pinocytosis: “cell drinking” similar event, but smaller in size and works on fluids. Sometimes these vesicles get transported to the opposite cell surface and get released – transcytosis. Exocytosis: Moving out Exocytosis is triggered in many cells by transient increase in cytosolic Ca 2+ Occurs via two pathways: Constitutive secretion, when release happens immediately after synthesis (e.g collagen fibers for ECM) and Regulated secretion, when release happens in response to outside signals (e.g. digestive enzymes). Portions of cell membrane become part of endocytotic vesicles during endocytosis; during exocytosis, it is returned to the cell surface. This process is called membrane trafficking. Signal Reception & Transduction Cells in a multicellular organism communicate to regulate tissue and organ development, to control their growth and division, and to coordinate their functions. Adjacent cells form gap junctions. Those located further away use receptors to detect and respond to various stimuli, with further set of cytoplasmic receptor proteins that produce complementary actions in a specific, programmed way. Cells with receptors for specific ligand (transmitter) are called target cells. Types of signaling include: Endocrine signaling when signal molecules (hormones) are carried in the blood from their sources to target cells. Paracrine signaling, chemical ligand diffuses in extracellular fluid, gets rapidly metabolized and effects only local target cells. Autocrine signaling, signals bind receptors on the same cells that produce them. Juxtacrine signaling, signaling molecules are cell membrane–bound proteins and need contact with target cells. Synaptic signaling A special type of of paracrine interaction, neurotransmitters act on adjacent cells throughspecial contact areas called synapses. This type of signaling is used by nerve endings and is very rapid and effective.  Receptors Receptors for hydrophilic signaling molecules, such as polypeptide hormones and neurotransmitters, are usually transmembrane proteins in the plasmalemma of target cells. They are classified in three groups: Channel-linked receptors, open channels to let the molecules through. Enzymatic receptors, which induce catalytic activity in associated peripheral proteins. G protein–coupled receptors, which stimulate G proteins, which then bind to GTP and other intracellular enzymes. This whole process is referred as signal transduction, in which ligands can be considered primary (first) messengers, which activate enzymes inside the cells. One such enzyme is adenyl cylcase, which generates large quantities of secondary messengers, some of which are cyclic adenosine monophosphate (cAMP), 1,2-diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP3). CYTOPLASMIC ORGANELLES: Ribosomes Inside cells we have organelles, which may be membranous (such as mitochondria) or non-membranous protein complexes (such as ribosomes and proteasomes) Ribosomes are 20×30nm basophilic macromolecules made of rRNA (ribosomal). Function is to assemble proteins from amino acids carried by tRNA (Transporter), dictated by mRNA (messenger). Consists of two sub-units (30 and protein sub-units). The ribosomes and their proteins are assembled in the nucleus. Sometimes multiple of them bind same strand of mRNA and form complexes polyribosomes or polysomes. Rough Endoplasmic Reticulum Is a membranous network found in most cells. Extends from surface of the nucleus throughout the cytoplasm and creates communicating channels cisternae (reservoirs). It’s surface is up to 30x of plasma membrane and is a major site of biosynthesis of proteins and lipids. Polyribosomes attached to some areas create rough and smooth ER. RER is prominent in cells that specialize in protein secretion. It has a highly regulate system to prevent nonfunctional proteins being forwarded to the pathway for secretion or to other organelles. Those that can’t be assembled undergo ER-associated degradation (ERAD) This “Quality control” is very important as it’s defect can lead to diseases, such as osteogenesis imperfecta, when bone cells synthesize and release defective procollagen fibers that can not assemble correctly and produce very weak bone tissue. Smooth Endoplasmic Reticulum Regions of that lack polyribosomes make up the (SER), which is continuous with RER, but is less abundant and not basophilic therefore can be viewed on TEM. SER has three main functions: 1. Synthesis of phospholipids and steroids which then get transferred to other membranes. 2. Contains enzymes such as cytochrome P450 family that detoxification of potentially harmful molecules. 3. SER vesicles are also responsible for controlled release of Ca2+ which allows cells for rapid response, particularly well developed in muscle cells (sarcoplasmic reticulum) where it’s needed for contraction. Golgi Apparatus A dynamic organelle also called Golgi complex completes posttranslational modifications of proteins produced in the RER and then packages and addresses these proteins to their proper destinations. Material moves to Golgi apparatus via transport veiscles, merges with it’s cis face and gets carried to it’s trans face where assambled products accumulate and eventually release. This forward movement of vesicles is mediated by coat protein COP-II while retrograde is mediated by COP-I. After transport the material get’s stored in secretory granules until their release by exocytosis.  Lysosomes Sites of intracellular digestion and turnover (reuse) of cellular components. Contain about 40 different hydrolytic enzymes and are particularly abundant in cells with great phagocytic activity (neutrophils, macrophages) and are able to break down macromolecules. The enzyme types vary from cell to cell, but most contain acid hydrolyases such as proteases, nucleases, phosphatase, phospholipases, sulfatases, and β-glucuronidase. Cytosolic components are protected from these enzymes by the membrane surrounding lysosomes and because the enzymes have optimal activity at an acidic pH (~5.0). Any leaked lysosomal enzymes are practically inactive at the pH of cytosol (~7.2) and harmless to the cell. Once lysosome fuses with the vesicle that has entered vie endocytosis it forms heterolysosome, which is larger and has heterogenous appearance due to its’ contents. After which indigestable material is left as residual body. Some long-lived cells accumulate as lipofuscin age pigment. Proteasomes Very small abundant protein complexes not associated with membrane, each approximately the size of the small ribosomal sub-unit. Their function is to degrade denatured or otherwise nonfunctional polypeptides. They also remove proteins no longer needed by the cell and provide an important mechanism for restricting activity of a specific protein to a certain window of time. Cylindrical structure made of four stacked rings, each composed of seven proteins including proteases. At each end of the cylinder is a regulatory particle that contains ATPase and recognizes proteins with attached molecules of ubiquitin, an abundant cytosolic 76-amino acid protein found in all cells. Failure of proteasomes to function can cause an accumulation of defective proteins and eventually damages nervous system (Alzheimer and Huntington disease) Mitochondria Glycolisis, an anaerobic conversion of glucose into pyruvate happens in the cytoplasm. While the mitochondria is a membrane-enclosed organelle, that contain arrays of enzymes that specialize in aerobic respiration and ATP production from pyruvate that was produced by glycolisis. This process yields about 15 times more ATP than anaerobic breakdown. Furthermore, some of the energy released in mitochondria does not generate ATP, but created heat for maintaining body temperature. They are elongated structures with diameters of 0.5-1 μm and lengths up to 10 times greater. Highly plastic and can change shape, fuse with each other, divide, and move as needed. Their numbers depend on how much energy does the cell or part of the cell needs (cardiac muscle has a lot).  Mitochondria Usually big enough to be visible with light microscopy, but TEM reveals two separate and different membranes which create inner matrix and intermembrane space. Outer membrane has multiple transmembrane proteins porins that form channels that allow passage of molecules. The inner membrane: possesses long folds called cristae, which increase the surface area. Their numbers also correspond to the amount of energy consumption of the cell. Enzymes for the electron-transport chain are embedded in the inner membrane. Another role of mitochondria occurs at times of cell stress, when the protein cytochrome c is released from the inner membrane’s electron transport chain. In the cytoplasm it activates set of proteases that that degrade all cellular components in a process called apoptosis. Mitochondrial DNA Unlike most organelles mitochondria are partly autonomous of nuclear genes and activities. They contain a small circular chromosome of DNA, ribosomes, tRNA and mRNA, very similar to bacteria. But they synthesize only small amount of local proteins. Some mutations in these genes can cause rare diseases, such as Myoclonic epilepsy with ragged red fibers (MERRF) is a rare disease occurring in individuals in whom cells of specific tissues, notably regions of skeletal muscle, inherit mitochondrial DNA with a mutated gene for lysine-tRNA, leading to defective synthesis of respiratory chain proteins which can produce structural abnormality in muscle fibers and other cells. Peroxisome Spherical and enclosed by single layer membrane. Produces enzymes that degrade H2O2 (Hydrogen peroxide) which can damage cell. Peroxisome catalases break down peroxides and also inactivate some toxic substances (liver and kidney cells). Cytoskeleton, Intermediate filaments, Cytoplasmic inclusions Nucleus Used literature Junqueira’s Basic Histology 15th ed. 42-51; 53-58 BRS Cell Biology Histology 8th ed. Cytoskeleton Complex array of: microtubules, microfilaments (actin filaments) and intermediate filaments. Can only be seen via electronic microscope. Determine shape of cells, play an important role in the movement of organelles and vesicles and even whole cells. Microtubules Organized in larger, more stable arrays called axonemes in the cytoplasmic extensions called cilia (“hair” almost all cells have one) and flagella. Microtubules are hollow, with an outer diameter of 25nm and a wall 5nm thick, which helps maintain cell shape. Can be linked by various proteins. Length is dynamic and variable and can be many micrometers long. The microtubules are polymers and are made of α and β tubulins (+&-). Their polymerization (assembly) is directed by microtubule organizing centers (MTOCs). This process happens when Ca and Mg Ions and various microtubule associated proteins are present. The dominant MTOC in most cells is the centrosome, it is organized around two cylindrical centrioles, each about 0.2μm in diameter and 0.3-0.5μm in length. Centrioles Each centriole is composed of nine highly organized microtubule triplets. Before cell division, specifically during the period of DNA replication, centrosomes duplicate and each centrosome has two pairs of centrioles. During mitosis centrosome divides in halves and each moves to the opposite end of the cell and become organizing centers for the of the mitotic spindle. Microtubule transport Microtubules also form part of the system for intracellular transport of membranous vesicles, macromolecular complexes, and organelles. Some examples: axoplasmic (axon cytoplasm) transport in neurons, melanin transport in pigment cells, chromosome movements by mitotic spindle and vesicle movements. These transport is carried out by motor proteins and utilizes ATP. Kinesins carry material away from the MTOC near the nucleus toward the plus end of microtubules (anterograde transport), while dyneins carry material along microtubules in the opposite direction (retrograde transport). (Some viruses such as Herpes and Rabies abuse this system) This system also extends ER from the nuclear envelope to plasmalemma and moves vesicles to and through the Golgi apparatus. Some drugs that can disrupt activity of mitotic spindle can be used in chemotherapy (vinblastine, vincristine, paclitaxel) Microfilaments (Actin Filaments) Abundant in all cells. Composed of actin sub-units and allow contraction of cells. Associated with myosin protein family. 5-7nm in diameter, polarized polymers, shorter and more flexible than microtubules, assemble in presence of K and Mg. Like microtubules actin filaments are highly dynamic. Monomers are added rapidly at the (+) or barbed end, with ATP hydrolysis at each addition; at the same time monomers dissociate at the (–) or pointed end. (exception myosin VI). This leads to migration of subunits through the polymer, which occurs rapidly in a process called treadmilling. Just as the molecular motors kinesin and dynein move structures along microtubules, various myosin motors use ATP to transport cargo along actin. Actin-myosin interactions are needed for: Transport of organelles, vesicles, and granules in the process of cytoplasmic streaming. Cytokinesis during cell splitting, endocytosis and forceful muscle contrations. Intermediate Filaments Intermediate in size between the other two. More stable and allows for mechanical stability of the cell. Some more specialized intermediate filaments include: Keratins: cytokeratins. Has acidic and basic forms, in all epithelial cells. Produce filaments with different chemical and immunologic properties. Some form large bundles (tonofibrils) and attach to certain junctions between epithelial cells. In some epithelial cells (skin) keratin accumulates (keratinization) and produces outer layer of dead cells for protection. Also produces various hard protective structures of skin, such as nails (as well as feathers, beaks, horns, and the scales of reptiles). Intermediate Filaments Vimentin: most common class III intermediate filament protein and is found in most cells derived from embryonic mesenchyme (connective tissue). Important proteins include desmin found in muscle cells and glial fibrillar acidic protein (GFAP) common in astrocytes (CNS supporting cells). Neurofilament: proteins of three distinct sizes make heterodimers that form the sub-units of the major intermediate filaments of neurons. Lamins: present in the cell nucleus, where they form a structural framework called the nuclear lamina just inside the nuclear envelope. Inclusions Are accumulated metabolites or other substances but have little to no metabolic activity. Most are transitory and have no membrane. Commonly seen variants are: Lipid droplets: accumulations of lipid filling adipocytes (fat cells) and present in various other cells. Glycogen granules: aggregations of glucose polymer, common in hepatocytes. Melanin: dark brown granules which in skin serve to protect cells from UV radiation. Lipofuscin: age pigment. Pale brown granule found in many cells, especially in stable nondividing cells (eg, neurons, cardiac muscle), containing a complex mix of material partly derived from residual bodies after lysosomal digestion. Hemosiderin: a dense brown aggregate of denatured ferritin proteins with many atoms of bound iron, prominent in phagocytic cells of the liver and spleen, where it results from phagocytosis of red blood cells. Nucleus; Nuclear envelope Nucleus is main structure of cell as it contains DNA (a code for all the cell proteins). Components include: Nuclear envelope, chromatin, nucleoli. Envelope: Selectively permeable barrier, between the nuclear and cytoplasmic compartments. Electronic microscopy shows 2 membranes, separated by 30-50nm perinuclear space. Outer membrane is continuous with RER, while Inner membrane is closely associated with meshwork of proteins - nuclear lamina (for stabilization). Major components of this layer are intermediate filament proteins called lamins. These two layers are bridged by 3000-4000 nuclear pore complexes made of nucleoporins. Proteins have specific import and export sequences that bind to transport proteins (importins, exportins), which in turn interact with pore complex proteins and use GTP. Chromatin Consists of DNA and all the associated proteins involved in its organization and function. Divided into 23 pairs of chromosomes (except gametes), each consisting of 2 identical chromatids connected by cohesin proteins. DNA is approx. 2m long with 3.2 billion base pairs and must be packaged in nucleus. This is done firstly by wrapping 150bp of DNA around the histone proteins and forming nucleosome, which has 4 histone paiir core (H2A,H2B,H3 and H4) and H1 outside. In electronic microscope nucleosomes an 50-80bp linker DNA (between them) have “beads on a string” appearance. They are dynamic, and their rearrangement allows DNA to be “unwrapped” and transcribed. Chromatin DNA in nucleosomes is further coiled with multiple steps and become visible via light microscopy during mitosis. 2 types of chromatin exist, euchromatin and heterochromatin. First being the gene rich “open” version of chromatin, that can be easily transcribed – “gene rich”, while heterochromatin has little to no transctiptional activity and is “gene poor”. Two types of heterochromatin can be observed in the chromosomes: Constitutive – mainly similar in all cells and contain repetitive sequences (centromeres, telomeres). While facultative is inactivated but can be “opened” and transcribed if necessary. Chromatin The ratio of heterochromatin to euchromatin seen with nuclear staining can provide a rough indicator of a cell’s metabolic and biosynthetic activity. Euchromatin is abundant in active cells – neurons, while cells with little synthetic activity, like circulating lymphocytes, have more heterochromatin. Facultative heterochromatin also occurs in small, dense “sex chromatin” or Barr body which is one of two X chromosomes present only in females. Recent studies have shown that heterochromatin tends to be located near the nuclear lamina, and that different chromosomes occupy different chromosomal territories, with more active domains located deeper. Karyotype is a microscopic image of chromosomes that allows to see any numerical abnormalities. Nucleolus A generally spherical, highly basophilic subdomain of nuclei in cells actively engaged in protein synthesis. This basophilic nature comes from high amounts of rRNA that is transcribed, processed, and assembled into ribosomal sub-units. Common cells with intensive protein synthesis and therefore high rRNA demand. The rRNA sub-units are matured and quickly associated with proteins, after which newly organized sub-units are exported in the cytoplasm via nuclear pores.  Cell cycle, Mitosis, Meiosis, Apoptosis; Stem cell and tissue renewal Used literature Junqueira’s Basic Histology 15th ed. 59 - 69 BRS Cell Biology Histology 8th ed. 44 - 52 Cell cycle Before differentiation, most cells undergo repeated cycles of macromolecular synthesis (growth) and division (mitosis). These events are called cell cycle. It has 4 phases: Mitosis – Cell division G1 – Time between mitosis and beginning of DNA replication S - DNA synthesis G2 – Time between DNA duplication and the next mitosis Cell cycle: Phases The G1 phase, usually the longest part of cycle, is a period of active RNA and protein synthesis. Also cell volume, halved from mitosis, returns to its previous size. The S phase is characterized by DNA replication, histone synthesis, and the beginning of centrosome duplication. In the relatively short G2 phase, proteins required for mitosis accumulate. As new postmitotic cells specialize and differentiate, cell cycle activities may be temporarily or permanently suspended, with the cells sometimes referred to as being in the G0 phase. Cell cycle activation Cycle is activated in postmitotic G0 cells by protein signals from the extracellular environment called mitogens or growth factors that bind to cell surface receptors and trigger a cascade of kinase signaling in the cells. After this cells stay at the beginning of S phase (restriction point), until enough material is gathered for S phase and DNA replication. Throughout the cycle there are several checkpoints that monitor of the cell is ready to progress. Overall this is regulated by a family of cytoplasmic proteins cyclins. Different cyclins are present at different stages of cell cycle and each activated one or more specific cyclin-dependent kinases (CDKs). Kinases then phosphorylate specific proteins transcription factors that express specific genes and enzymes of specoific cytoskeletal sub-units, triggering the next phase of the cell cycle. As one phase ends the proteins associated get ubiquitinated and removed rapidly by proteasomes and a new cyclin that promotes activities for next phase takes over. Cell cycle activation Progression through cell cycle is halted by harmful conditions such as inadequate nutrition (nutrient stress), inappropriate cellular microenvironments, or DNA damage, the last one being most important, since DNA damage can stop cycle at any stage. If nuclear DNA can not be repaired, tumor suppressor genes (TSP) are activated. Mutations are not always detected and corrected, and if such a change happens in a gene important for cell cycle activities (growth factor genes, their receptor genes or signaling kinase genes), cell cycle may be affected and growth may occur in a less-regulated manner. This is usually detected by the TSP such as p53. Failure to detect these will result in further changes and various types of cancer. Mitosis (1) Is period of cell division and only part of cycle distinguishable by light microscope. Cell divides into 2 daughter cells which receive identical copies of DNA. The periods between mitosis (G1, S and G2) are collectively called interphase. Has 4 phases: Prophase: Nucleolus disappears, chromatin condenses into chromosomes, 2 centrosomes (each with duplicated centrioles) separate and go to opposite poles. Lastly the nuclear lamina and nuclear pore complexes disassemble. Metaphase: Chromosomes condense further, protein complexes called kinetochores attach them to the mitotic spindle. Cell is spherical in shape and chromosomes are moved into alignment at the equatorial plate. Mitosis (2) Anaphase: Sister chromatids (now chromosomes) separate and move toward opposite spindle poles by a combination of microtubule motor proteins and dynamic changes in the lengths of the microtubules as the spindle poles move farther apart. Telophase: Chromosomes decondense (uncoil), spindle depolymerizes and nuclear envelope begins to reassemble around chromosomes. A belt-like contractile ring of actin filaments associated with myosins develops in the cortical cytoplasm at the cell’s equator. During cytokinesis at the end of telophase, constriction of this ring divides cytoplasm and its organelles are divided into two daughter cells. Most tissues undergo cell turnover with slow cell division and cell death. Nerve tissue and cardiac muscle are exceptions because their differentiated cells cannot undergo mitosis Stem cell and tissue renewal Many tissues and organs contain a small population of undifferentiated stem cells whose cycling serves to renew the differentiated cells of tissues as needed. Many stem cells divide asymmetrically, one daughter cell remains as a stem cell while the other becomes committed to a path that leads to differentiation. Most mitotic cells here are not stem cells but the more rapidly dividing progenitor cells or transit amplifying cells because they are in transit along the path from the stem cell niche to a differentiated state, while still amplifying by mitosis the number of new cells available for the differentiated tissue. In tissues with stable cell populations, such as most connective tissues, smooth muscle, and the cells lining blood vessels, stem cells are not readily apparent and differentiated cells undergo slow and episodic division to maintain tissue integrity. Meiosis Specialized process with two unique and closely associated cell divisions that occurs only in the cells that will form sperm and egg cells (gametes). Two key features characterize meiosis: 1) Homologous chromosomes of each pair (one from the mother, one from the father) come together in an activity termed synapsis. During this double-stranded breaks and repairs occur in the DNA, some of which result in reciprocal DNA exchanges called crossovers between the aligned homologous chromosomes. This produces new combinations of genes. 2) The cells produced are haploid, having just one chromosome from each pair present in the body’s somatic cells. The union of haploid eggs and sperm at fertilization forms a new diploid cell (the zygote) that can develop into a new individual. Important events of meiosis During greatly elongated prophase I (first division), partially condensed chromatin of homologous chromosomes begins to come together and physically associate along their lengths during synapsis. Because each of the paired chromosomes has two chromatids, geneticists refer to synaptic chromosomes as tetrads to emphasize that four copies of each genetic sequence are present. In human spermatogenesis prophase I normally lasts for 3 weeks; oocytes arrest in this meiotic phase from the time of their formation in the fetal ovary through the woman’s reproductive maturity, that is, for about 12 years to nearly five decades. Apoptosis Rapid, highly regulated cellular activity that eliminates defective/unneeded cells, by turning them into apoptotic bodies which quickly get phagocytosed. Apoptotic cells do not rupture and release their contents, unlike cells that die as a result of injury and undergo necrosis. This is highly significant because release of cellular components triggers a local inflammatory reaction and immigration of leukocytes. It’s controlled by cytoplasmic proteins in the Bcl-2 family, which regulate the release of death-promoting factors from mitochondria. Apoptosis changes (1) Activated by either external signals or irreversible internal damage, specific Bcl-2 proteins induce a process with the following features: Loss of mitochondrial function and caspase activation: Bcl-2 proteins associated with the outer mitochondrial membrane compromise membrane integrity, stopping normal activity and releasing cytochrome c into the cytoplasm where it activates proteolytic enzymes called caspases. The initial caspases activate a cascade of other caspases, resulting in protein degradation throughout the cell. Fragmentation of DNA: Endonucleases are activated, which cleave DNA between nucleosomes into small fragments. Shrinkage of nuclear and cell volumes: Destruction of the cytoskeleton and chromatin causes the cell to shrink quickly, producing small structures with dense, darkly stained pyknotic nuclei that may be identifiable with the light microscope. Apoptosis changes (2) Cell membrane changes: The plasma membrane of the shrinking cell undergoes dramatic shape changes, such as “blebbing,” as membrane proteins are degraded and lipid mobility increases. Formation and phagocytic removal: Membrane-bound remnants of cytoplasm and nucleus separate as very small apoptotic bodies. Newly exposed phospholipids on these bodies induce their phagocytosis by neighboring cells or white blood cells. Epithelial tissue Used literature Junqueira’s Basic Histology 15th ed. Pages 71 - 95 (Chapter 4) BRS Cell Biology Histology 8th ed. Pages 66 – 84 (Chapter 3) Four basic types of tissues Despite the complexity, human body only has 4 basic types of tissues: Epithelial, Muscular, Nervous and Connective A tissue is group of cells, performing similar functions. All basic tissues have ECM (Extra cellular matrix), however connective tissue has more than others. Organs are composed of combination of these tissue types and therefore they are divided into parenchyma (functional part) and stroma (supportive part). Stoma is always made of connective tissue, except for the CNS. General information about epithelia Composed of closely aggregated and adhered polyhedral cells and thin layer of ECM. They line cavities of organs and cover the body syrface. Therefore any substances that enter/ exit body must cross epithelia. Their main functions are Covering/ lining/ protecting (epidermis), absorption (intestinal lining), Secretion (parenchyma of glands). Some epithelium can contract (myoepithelia) or can be sensory (taste buds, olfactory epithelium) Characteristic features of epithelial cells Epithelia can be classified by shape: Squamos (flat), Cuboidal (cube shaped), Columnar (tall). Shape of nuclei also follow cell shapes. Transitional epithelium is rare and has multiple cell shapes And by the amount of layers: Simple (one), Stratified (many), Pseudostratified (only appears as multilayered but is one layer) Characteristic features of epithelial cells Most epithelia are near connective tissue which has good blood supply which gives them oxygen and nutrients, therefore even thick epithelia don’t usually contain blood vessels. The connective tissue under the epithelia is called lamina propria, and the contact area between them is increased by small evaginations - papillae. Polarity Epithelial cells generally show polarity, which means that organelles and membrane proteins are distributed unevenly within the cell. Because of this property, these cells have basal pole – contacting the connective tissues udnerneath, apical pole – opposite end usually facing the space. And some also have lateral surfaces which connects two cells to each other.  Basement membranes The basal surface of epithelia rests on a thin extracellular sheet of macromolecules – basement membrane. Is semipermiable and can be seen with light microscopy, but it has two parts which can only be visualized by electronic microscopy: basal lamina – nearest to the epithelia and more diffuse reticular lamina. Terms: basal lamina and basement membrane are sometimes used as same. Basal lamina contains molecules secreted from basal poles: Type IV collagen - which self-assembles into a net, Laminin – are large glycoproteins (glucose + protein) which attach integrin proteins in the basal cell membrane, Nidogen and Perlecan – connect laminin and type IV collagen mesh. Reticular lamina contains type III collagen which is connected to basal lamina via type VII collagen. The function of basement membranes is structural support, filter and attachment. U Intercellular adhesion and Junctions There are several membrane-associated structures that provide adhesion and communication between cells, epithelia has lots of such stuctures, because they need to adhere tightly to other cells an basal laminae, especially in areas subject to friction and other mechanical forces. Types of epithelial junctions: Tight or Occluding junctions: Form a seal between adjacent cells. Adherent or anchoring junctions: Are sites of strong cell adhesion. Gap junctions: are channels for communication between adjacent cells. Tight junction are also called zonnula occludens, which completely encircles the cell and is formed by right interactions of proteins claudin and occludin. This junctions restrict movements of membrane lipids from the apical to the basal side of the cells and vice versa, therefore we get two domains, apical and basolateral. U Intercellular adhesion and Junctions Adherens junction or zona adherens also encircles whole cell, and anchores whole cell to it’s neighbors. This action is mediated by cadherins, transmembrane glycoproteins that bind each other in the presence of Ca2+. Another junction is desmosome or macula adherens which attache cells to each other via their cytoskeletons Hemidesmosomes attach cells to basement membrane. Gap junctions mediate intercellular communications, they work via connexin proteins and let the messenger molecules through. Apical cell surface specializations Columnar and cuboidal epithelial cells may have specialized projections from the apical poles: microvilli, stereocillia and cillia. Microvilli are cytpolasmic projections seen with electronic microscope. Have variable shape, length (0.1-1um) and numbers and increase surface area for better absorption. These structure is called brush or striated border of the cell. Stereocilia are much less common, also seen in absorprive epithelia, but additionally they can be found in sensory organs for motor detection. (Inner ear, vestibular apparatus) Cillia are long and highly motile. Additionally almost all cells have at least one non motile primary cillium, which is enriched with receptors for detection of light, motion and flow of liquid past the cells. Types of epithelia The stratified squamous keratinized epithelium covers mainly skin and prevents dehydration. During keratinization the cuboidal cells at the base flatten, accumulate keratin and become inactive packets (also loose nuclei) Internal organs are covered by stratified squamous nonkeratinized epithelia. Stratified cuboidal and columnar are rare and line ducts of glands and eyelid conjunctiva. Transitional epithelium or urothelium can be found in urinary tract, where it’s top layer of umbrella cells protect underlying tissues from hypertonic and potentially cytotoxic effects of urine. U Secretory Epithelia & Glands Secretory cells may synthesize, store, and release proteins (eg, in the pancreas), lipids (eg, adrenal, sebaceous glands), or complexes of carbohydrates and proteins (eg, salivary glands). Some glands secrete mostly water and electrolytes (ions) – Sweat glands. Simple cuboidal, columnar and pseudostratified epithelia often have unicellular secretory glands – goblet cells of in small intestine which secrete mucus. After development, exocrine glands remain connected with the surface and secrete outside the body or in cavities. While endocrine glands loose the connection, therefore lack ducts and secrete directly into blood. Secretory Epithelia & Glands Types of secretion Merocrine secretion: This is the most common method of protein or glycoprotein secretion and involves typical exocytosis from membrane-bound vesicles or secretory granules. Holocrine secretion: Here cells accumulate product continuously as they enlarge and undergo terminal differentiation, culminating in complete cell disruption that releases the product and cell debris into the gland’s lumen. This is best seen in the sebaceous glands producing lipid rich material in skin Apocrine secretion: Here product accumulates at the cells’ apical ends, portions of which are then extruded to release the product together with small amounts of cytoplasm and cell membrane. Lipid drop lets are secreted in the mammary gland in this manner Types of secretion Connective Tissue BRS Cell Biology Histology 2018 8th ed. Pages 85 – 97 Junqueira’s Basic Histology Text and Atlas 2018 15th ed. Pages 96 – 120 Overview Formed primarily of extracellular matrix (ground substance, fibers and cells) Has multiple functions: 1. Supports organs 2. Acts as a medium for exchange 3. Protects against microorganisms 4. Mounts immune response 5. Repairs damaged tissues 6. Stores fat Cells Grouped in 2 categories: Fixed cells that originate in connective tissue and remain here Transient cells that originate somewhere else and remain in connective tissues temporarily. Fixed cells: Fibroblasts Predominant cells in connective tissue proper Arise from mesenchymal cells of the embryo Often possess an oval nucleus with two or more nucleoli (Produces ribosomes) Seldom undergo mitosis except in wound healing and may differentiate into adipose or chondrocytes Responsible for manufacturing almost the entire ECM When synthesizing it, the become fusiform (spindle-shaped), have well-developed RER and many Golgi complexes and are called active fibroblasts Inactive forms are referred as fibrocytes Fixed cells: Pericytes Also known as adventitial cells or perivascular cells Associated with capillaries, share their basement membranes Embryonic mesenchymal cell derivatives and may retain a pluripotential capacity Have characteristics of endothelial cells as well as of smooth muscle, as they contain actin, myosin, and tropomyosin and assist in influencing blood flow May differentiate into fibroblasts, smooth muscle cells, endothelial cells of vessel walls Fixed cells: Adipose cells (adipocytes) Arise from mesenchymal cells and are surrounded by a basal lamina Do NOT normally divide beyond age 2 years, except for morbid obesity Function, synthesis, storage, and release of fat as well as for releasing hormones Two types: Unilocular, have a single large fat droplet that squeezes the cytoplasm and nucleus. Has insulin, GH, norepinephrine and glucocorticoid receptors. Multilocular, smaller, store fat in many small droplets. Central, spherical nucleus. Generate heat. Fixed cells: Mast cells Arise from myeloid stem cells in bone marrow and reside near small vessels Among the largest cells of connective tissue proper. Central spherical nucleus; cytoplasm filled with granules full of primary mediators Well-developed Golgi complex, scant RER, and many dense granules Two populations: connective tissue mast cells (contain heparin) and smaller mucosal mast cells, located in the mucosa of the alimentary canal and of the respiratory tract (contain chondroitin sulfate). None in CNS to prevent inflammation Activated mast cells convert phospholipids in their cell membranes into arachidonic acid by the enzyme phospholipase A2 which is then converted into secondary mediators such as leukotrienes and Mast cell immune response Mediate immediate (type I) hypersensitivity reactions (anaphylactic reactions) Sensitization occurs, when an individual is exposed to a specific allergen for the first time plasma cells manufacture immunoglobulin E (lgE), which bind to mast cells and basophils. During a second exposure to the same allergen, it allergen binds to the membrane-bound IgE, leading to clustering of the allergen-lgE complexes which triggers primary and secondary mediator release from the mast cell. Examples: Hay fever Asthma Anaphylactic shock Fixed/Transient cells: Macrophages Both fixed and transient Liver Kupffer cells; Lung dust cells; Bone osteoclasts; Skin Langerhans cells; and CNS microglia are all macrophages. Phagocytes of connective tissue proper Originate in the bone marrow as monocytes, migrate and differentiate. Colony-forming unit monocyte factor (CFU-M) facilitates differentiation, while macrophage colony-stimulating factor (M-CSF) promotes mitosis. They are antigen-presenting cells (eat, break down and place epitopes on surface) Also destroy large foreign particles and bodies, can also form foreign body giant cells Transient cells: Lymphoid cells Arise from lymphoid stem cells during hemopoiesis Specialized in mounting an immune response T lymphocytes (T cells) initiate the cell-mediated immune response B lymphocytes (B cells) differentiate into plasma cells, which manufacture antibodies and function in the humoral immune response Natural killer cells (NK cells) display cytotoxic activity against tumor cells Transient cells: Plasma cells Antibody-manufacturing cells that arise from activated B lymphocytes Eccentric nucleus possessing clumps of heterochromatin. Deeply basophilic cytoplasm due to abundance of RER Area adjacent to nucleus is pale and has Golgi complex (negative Golgi image) Most abundant at wound entry sites or in areas of chronic inflammation Transient cells: Granulocytes Have cytoplasmic granules, at sites of inflammation leave blood and enter tissues Arise from myeloid stem cells 3 types: Neutrophils: Digest bacteria and form pus. Eosinophils: follow eosinophil chemotactic factor, bind to parasites and release cytotoxins. Then release enzymes that cleave histamine and leukotriene C, thus moderating the allergic reaction. Basophils: similar to mast cells but they circulate in blood Classification: Embryonic Classification of connective tissue is based on cells/fibers proportion and on the arrangement and type of fibers Mucous tissue (Wharton jelly): jelly-like matrix, some collagen fibers and large fibroblasts Mesenchymal tissue: almost exclusively in embryos. Has a few scattered reticular fibers, in which star-shaped, pale-staining mesenchymal cells are embedded Classification: Connective tissue proper Types: Loose (areolar): well vascularized, flexible, and not very resistant to stress. Fewer fibers, and more cells. Predominant connective tissue of the hypodermis. Dense: more fibers (Type I collagen) but fewer cells. Can be irregular (dermis, organ capsules) and regular (Collagenous: tendons and ligaments. Elastic ligamentum nuchae, ligamentum flava) Elastic: branching elastic fibers (elastin and fibrillin). Present in dermis, lungs, elastic cartilage/ligaments and in large (conducting) vessels as fenestrated sheaths Reticular: type III collagen, liver sinusoids, smooth muscle, fat cells, stroma of lymphatic organs, bone marrow, endocrine glands and the reticular lamina Adipose tissue: discussed in the next slides. Connective tissue proper: White adipose tissue 3 types of cells: white, beige, and brown Rich neurovascular supply, stores fat, cushions body, brown cells generate heat White adipose tissue: Almost all of the adult adipose tissue. Consists of unilocular cells VLDLs in the capillaries of adipose tissue get absorbed and stored as triglycerides By influence of insulin, can also synthesize fatty acids from glucose and amino acids Neural impulses/adrenaline can cause release of stored fat by hormone-sensitive lipase Is an endocrine organ: Leptin adiponectin, resistin, and retinol-binding protein-4 – By adipocytes TNF-cx, interleukin-& [IL-6], and adipocyte fatty acid-binding protein – By macrophages Connective tissue proper: Brown adipose tissue Multilocular cells Contain many large mitochondria Located along the vertebral column, above kidneys, and supraclavicular region Generate heat: Thermogenin a a transmembrane protein in mitochondria uses ATP Found in infants (also in hibernating animals) Beige adipose cells: intermingled with white adipocytes, especially in the inguinal region