PHA206 Cytology Introduction: Cell Structure & Function

PHA206 General Cytology

• The course aims to cover the structure and function of eukaryotic cells, tissue classification, cell injury and death, and the relationships between cytology, histology, physiology, pathology, and therapeutics.
• It will help recognize histological specimens and microscopic structures and analyse tissue relationships within organ systems.

Cytology Introduction

• Cytology studies cells, their origin, structure, function, and pathology.
• A cell maintains its structure via a chemical system and is the smallest unit to perform life processes; metabolism, regulation, locomotion, synthesis, transport, respiration, nutrition, growth, and excretion.

History

• Cells remained unseen until the invention of the microscope, which allowed Hooke (1665) and van Leeuwenhoek (1675) to draw the first cells.
• Cell theory, which states that cells only come from other cells, emerged around 1840, contradicting spontaneous generation.

Cell Theory Principles

• All living things consist of one or more cells.
• Cells are the basic units of structure and function in an organism.
• Cells originate only from pre-existing cells through replication.

Biological Organization

• Life is organized into levels; unicellular organisms function independently, while multicellular organisms' cells coordinate at five levels.
• Cells form tissues, tissues form organs, organs form organ systems, and organ systems form, organisms.

Cell Size

• Most cells are small with the human egg being an exception and being the largest cell
• Cell size is limited by nucleus control in relation cytoplasm volume and a surface area to volume ratio.
• Smaller cells have a relatively larger surface area for nutrient and material exchange.

Cell Shape

• Cell shapes vary based on function with neurons that carry long-distance electrical messages having a slender process shape.
• Photoreceptor cells contain light-absorbing molecules in a specialized outer segment, which is a modified cilium.
• Immune cells and Lymphocytes alter their shape to move through tissues with Lymphocytes squeezes to move between tightly packed cells.
• Red blood cells have a biconcave shape to enhance oxygen diffusion

Cell Classification

• Cells fall into two main classes: prokaryotic and eukaryotic.
• Prokaryotic cells lack a nuclear envelope and are generally unicellular bacteria.
• Eukaryotic cells possess a nucleus where DNA (deoxyribonucleic acid) is separated from the cytoplasm.

Prokaryotes

• Prokaryotes are widespread, adaptable organisms which able to thrive in extreme conditions such as high temperatures (up to 130°C) or a lack of oxygen.
• They reproduce rapidly and form resistant spores for survival and possess one of three shapes:
  • Spherical (cocci).
  • Rod-like (bacilli).
  • Helically coiled (spirilla),
• Prokaryotes are single-celled, lack a membrane-enclosed nucleus.

Prokaryote Structure Overview

• Most have a rigid cell wall, a cell membrane and cytoplasm.
• The ~70A-thick cell membrane is also known as the plasma membrane.
• They range in size from 1 to 10 picometers.
• Their cytoplasm includes a single chromosome (DNA), RNA, enzymes, and ribosomes.
• Some bacterial cells have flagella for locomotion and pili, which can act as conduits for DNA during conjugation or aid in attachment to a host.

Prokaryote Classification

• Classification systems divide prokaryotes into prokaryotae kingdoms, also known as monera, and divide them into Cyanobacteria and bacteria
• Morphology, biochemical and molecular traits further classify bacteria
• Mycoplasmas that are of a prokaryotes type lack a rigid cell wall, are the smallest of all living cells, with sizes as small as 0.12 μm

Prokaryote Structure & gram Staining

• Some bacteria are classified based on the properties of cell walls
• Gram-positive bacteria, and gram-negative bacteria as well as an outer membrane surrounds the cell wall that excludes gram stain
• Gram staining began in 1884 by Christian Gram and involves heat treatment of fixed cells and staining with crystal violet and iodine and destaining with ethanol or acetone
• Gram-positive bacteria specifically, are devoid of the outer membrane

Eukaryotes

• Eukaryotic cells range from 10 to 100 picometers in diameter and posses membrane-enclosed organelles.
• Eukaryotic cells are structurally and functionally complex with organization at all levels.
• They are divided into the protozoa (unicellular) and metazoa (multicellular) groups.
• Their complexity helps with competitive advantage in environments with limited resources.

Viruses

• Viruses must adsorb to the host cell, inject viral chromosome into the host cell, which then replicates before encapsulation and then releases more viruses.

Biomembranes

• Cell membranes consist of a phospholipid bilayer with peripheral (surface) or integral (spanning) proteins.
• Each phospholipid layer includes a hydrophilic phosphate end and a hydrophobic lipid tail.
• Membrane components are constantly moving known as the Fluid Mosaic Model.

Biomembrane permeability

• Biological membranes selectively regulate substance passage.
• They contain receptors and channels for specific molecules like ions, nutrients, wastes, and metabolic products.
• Organelle membranes separate vital but incompatible metabolic processes, like the mitochondrial membrane separating energy production by ATP synthesis.

Lipid Bilayer Composition

• Membranes are mainly composed of a lipid bilayer containing phospholipids, cholesterol, and glycolipids.
• The lipids determine the membrane's form (sheets or vesicles) and give it a fluid characteristic that allow permeation of fatty acids but repelling large, water-soluble molecules.

Protein in Membranes

• Embedded proteins in membranes play roles including transport (ions, water-soluble molecules), channel formation (free diffusion) and cell recognition with carbohydrate molecules participating for various functions.

Secretory Granules

• These granules are condensing membrane bound vesicles from the Golgi apparatus.
• In cells they store products until exocytosis signals release like neurotransmitters at neuron terminal buttons.

Endosomes and Endocytosis

• Endosomes are small membrane-bound vesicles containing extracellular material.
• Endocytosis is the internalization of proteins and soluble macromolecules.

Lysosomes

• They are acidic membrane bound organelles containing a battery of degradative enzymes.
• They are variable in size, morphology, and are exclusively found in animal cells where they degrade obsolete cell components and materials from endocytosis or phagocytosis.

Lysosomes (Primary / Secondary)

• Primary lysosomes are spherical and contain no obvious particulates or debris.
• Secondary lysosomes are larger and irregularly shaped, appearing from a fusion of primary lysosomes with bound organelles and vesicles.

Peroxisomes

• These are organelles of spherical shape are in all animal cells, except erythrocytes.
• They contain oxidases to oxidize organic substances, forming hydrogen peroxide and contain the enzyme catalase.
• Energy from fatty acid oxidation is converted into heat with acetyl groups are transported for cholesterol synthesis.

Ribosomes

• Ribosomes appear in two basic types, which are the mitochondrial (like prokaryotic) ribosomes being relatively small at 20nm
• Cytoplasmic ribosomes of eukaryotes, are larger than the first type at 25nm
• They have 2 unequal ribosomal subunits with cytoplasmic ribosomes contain ribosomal RNA (rRNA), which is synthesized in the nucleolus.

Ribosomal Accumulations

• Light microscopy reveals ribosome accumulations as basophilic patches (ergastoplasm) in glandular cells and Nissl bodies in neurons.

Endoplasmic Reticulum (ER)

• The ER is the largest eukaryotic membrane enclosing sacs called cisternae and includes functions such as:
  • sER is used for hydrophobic chemical detoxification, along with lipid synthesis
  • rER is used for the synthesis of membrane proteins and secreted proteins
• The rER is abundant in cells synthesizing specific secreted proteins, such as neurons.

Golgi Apparatus

• A stack of flattened membrane vesicles or cisternae with spherical membrane-limited vesicles.
• Golgi cisternae are constructed of three regions: the cis, medial, and trans components.

Golgi network

• Two coated vesicle classes transport protein to and from the rER.
• The rER carries vesicles surrounded by COP-I and COP-II protein coat complexes.
• COP-II facilitates anterograde transport from the rER to the cis-Golgi network (CGN), while COP-I facilitates retrograde transport from the CGN back to the rER.

Proteasomes

• These are very small abundant protein complexes not associated with membrane, but of a similar size to ribosomal subunits for degrading denatured nonfunctional polypeptides.
• They recognize proteins tagged with ubiquitin molecules, with the ubiquitin being an abundant cytosolic protein

Cell Inclusions

• These are nonmoving and nonliving cytoplasmic or nuclear structures with staining properties, which are produced by cell metabolism.
• They may or may not be surrounded by plasma membranes.
• Lipofuscin is a brownish-gold pigment commonly seen in non-dividing cells from oxidative degradation and which accumulates during aging.
• Hemosiderin is an iron-storage complex resulting from hemoglobin breakdown with and presence relating to red blood cell removal.
• • Glycogen stores glucose, particularly in the liver and muscle
• Lipid inclusions are nutritive inclusions like fat droplets that are used for cellular metabolism found in adipocytes.

Nucleus

• The nucleus is composed of the nuclear envelope along with chromatin and a nucleolus.

Nuclear envelope

• These are double membranes which separate a narrow 30nm-50nm perinuclear space
• The inner nuclear membrane has a nuclear lamina with lamins, and bridges with protein and chromatin.

Nucleolus

• Nucleolus is typically sphere shaped that expresses basophilia and makes protein
• Basophilia has to do with highly concentrated ribosomal RNA which transcribes to process and is complexed into ribosomal subunits
• Regions containing rRNA genes assemble nucleoli which synthesis protein in growing or secreting cells

Mitochondria

• Mitochondria is a double membrane with a highly folded inner membrane called cristae for respiration. Two compartments are found; the intermembrane space with matrix including mitochondrial DNA, RNA and ribosomes.

Cytoskeleton

• A mesh of microtubules, microfilaments, and intermediate filaments.
• The cytoskeleton provides cell shape stability.
• It is important in cell movement to assist cytoplasmic components.

Microtubules

• These are thick elements of the cytoskeleton that have a diameter of 24 nm, that are made of tubulin heterodimers
• These are important with cellular shape, transporting in neurons, dispersion of melanin in pigment cells and movement of chromosomes
• Directed by organizing centers

polarized structures

• Microtubules are more easily polymerized at the side
• Microtubules are sensitive when it comes to instability and depolymerization
• Usually its located at MTOC in cells

Microtubules in cells

• They appear throughout the cytoplasm, with the centrioles, flagella, basal bodies and mitotic spindle apparutatus

Centrioles

• Centrioles are cylindrical organelles composed of nine microtubule triplets, 150 nm in diameter.
• Interphase cells contain perpendicular centriole pairs, surrounded by pericentriolar bodies.
• Cytoplasmic microtubules originate from pericentriolar bodies.
• Associated Golgi complexes constitute the cell cytocenter near the nucleus.

Transport between the Microtubules

• This is carefully controlled by motor proteins using ATP
• Kinesins moves MTOC closer to the nuclus helping anterograde transport
• Cytoplasmic dyneins go the opposite direction and assist retrograde transport to the nucleus,
• Important with sending ER away from the nuclear envelope by use of golgi apparatus.

Microfilaments

• Microfilaments are general submicroscopic organelles. There are three types of filaments
  • Myofilaments are of muscular tissue
  • Neurofilaments are of nerve cells
  • Tonofilaments are of epithelia

Motility

• Actin microfilaments aid cellular functions like motility and most functions
• The same amounts are added and monomers dissociate after reaching the opposite end
• Migration follows subunit migration through the polymer
• Cells locomotion is achieved by polymerization
• Cells locomotion is is from force and extensions of a crawling cell

Proteins and Cell motility

• Myosin proteins use tap to ship cargo along f-actin
• Cell movement is only going till barbed the positive end and myosin VI is the know protein that can act in another direction

Intermediate Filaments

• Intermediate filaments, ranging from 10-12 nm thick, are of a intermediate area between microtubules and microfillaments
• Keratin isoforms compose subunits

Filaments in cells

• Cytokeratins accumulate into an outerlayer of cells in the skin called keratinization
• Vimentin is popular type of filament found in mesenchyme
• Vimentin-like protein is found in muscle with desmin Glial fibrillar acidic

Intercellular Junctions

• Lateral surface of epithelial cells exhibit some junction
• Tiight and occluding junctions form a seal
• Anchoring junctions are a are for strong cell adhesion
• Gap junctions connect channels between

Histological and cytological assessment of specimens

• Preparation has to account for fixation, embedding, sectioning, staining and mounting.

Types of Specimens

• Collection of Specimen uses Exision specimens to remove tissues, while incisional biopsy removes tissue for diagnosis. Along with Punch biopsies, Shave biopsies, Core biopsies and Curettings

Specimen Collection

• Punch biopsies are to remove tissue from locations like the skin with specialized equipment
• Shave biopsies are use to shave tissue

Core Needle Biopsy

• This takes a needle to collect tissue from locations like the breast bone

Curettings

• These include removal of tissue from the uterus or cervix

Fixation process

• Its important to carry out fixation for autolysis and putrefaction
• Maintain cells and tissues for the best result

Types of Solutions for Fixation

• Formalin is commonly used with 40%. Glutarldehyde use for microscopy and osmium for best results

Cytological Preservation

• Alcohol and Carnoy's Fixative is what fixes results

Tisse Processing with Automation

• Automated machines that are responsible for dehydration and impregnation stages

Hydration process of sample

• Step 1 is dehydrating so water doesnt build up the the wax

The Steps of Rehydration

• Water molecules pass through with a grade of a high concentration of alcohol to stop wax

Clearing in automation

• An agent the has a role to remove alcohol from the tissue with a fluid the wax can take soluble action

Types of reagents for Clearing

• Xlylene most is common but with issues including being very toxic but other are still relevant including benzene, choloform

Embedding tissues with molds

• This process can be done with molds including blocks of tissues with number tags

Sectioning in microscope

• After excessive tissue get trimmed tissues can then be mounted for sectioning and use a blade called a cryostate with frozen material

Mounting process

• Section are obtained from knife that use forcp and placed into a slide after drying them with water

Staining for samples

• hematyoxin is popularly used and used an acid to show an acidic colour
• Eosin in the pposite a base the stains pink

Silver Staining

• Silver staining with reticular staining
• Nuclei would be colourless and the other would have a brown tinge

Types of staining for cells

• Orcein would have a elstaic colouration while Waigerts would cause different stains

Romanowsky for granules

• Stain to show granuales in blood
• For a pink stain you might find erythocytes while blue might get monoctyes

Prussian Blue Staining

• Important for finding a deomonstartiin iron on stain

Fat Staining

• Uses Sudan type is most used but oil red exists asweell

Histo/cytochemistry

• Locatization methods of certain constitutents and cells
• Can identify regeants as well
• Can idenfify by visual

Immnuo histo

• This uses antibodies to check for certian antigens
• Check samples tissue

Immunoperoxidase

• This has staining properties to idnetify the nature of Langerhans

Cytospectrum

• Methods need to be well thought out
• Spreading must be dont with care for platinum loop

Extoliative Cytology

• Branch of cytology thats studys cells from epitelial surfaces
• Study organ state by examining it
• Can find cancer signs using this and also using diagnostic cytology

Tissue Basics

• T issues are an organ that functions a certain way and there is four basic types

Epithliium

• Epithlium is charactreised by it's appearence at the epetheliam surface and classified by cellshape and layers