HISTOLOGY FULL-TEXT

Chapter 3 CYTOLOGY ll

A CELL MEMBRANE OR PLASMALEMMA

1 Although looking trilaminar in routine EM, it behaves as if it comprises a double layer of lipid molecules, in which proteins are distributed asymmetrically in a mobile mosaic pattern. Thus, some proteins span the width of the membrane, and may vary rates of transport by changes in their conformation. Others, as enzymes, receptors, or adhesion molecules, etc. have active domains at the surface held in correct position by intramembranous domains imbedded in the lipid layer, and intracellular domains to engage in events inside the cell.
A sometimes fuzzy-looking coat of glycoprotein - glycocalyx - sticks to the external face of the membrane.

2 Functions of the membrane are:

1. Firm attachment to other cells or a basal lamina; membrane specializations for this are: (a) junctional complexes, (b) gap junctions/ nexuses, (c) desmosomes, (d) hemi-desmosomes, (e) intercalated discs, and (f) membrane interdigitations; more details Chapter 4.D.l.
2. Movement of the cell itself by pseudopodial, filipodial, or lamellipodial extensions (think karate: fist, finger, or side of the hand) and the release of any firm attachments, or by flagellate activity, e.g., by sperm. (Microspikes and ruffles are alternative names for filopodia and lamellipodia, respectively.)
3. Movement of materials outside the cell by the activity of cilia, e.g., ciliated epithelia of the respiratory tract and uterine tube. The wide-spread occurrence of solitary cilia (flagella), e.g., on neurons, adrenal cells, smooth muscle, may involve a vestigial body or one still functional. The stereocilia of the male reproductive tract are non-motile, clumped, long microvilli, probably absorptive.
4. Transport of materials in and out of the cell served by: (a) permeability (selective) of the membrane, (b) active transport through the membrane, (c) endocytosis, and its more scaled-up forms - pinocytosis and phagocytosis, (d) exocytosis; and increased exchange surface area by (e) microvilli (thousands on a cell), and (f) infoldings of membrane.
5. Communication and transduction. Each cell collaborates with both adjacent cells, and those of the whole body, for development, growth, homeostasis, regeneration, and its own particular task. The importance of the cell membrane in receiving and sending the necessary signals is stressed by the number of examples given:
   (a) The binding of hormones to receptors on the membrane.
   (b) The binding of the lymphocyte's membrane receptor to an antigen.
   (c) Transmitter chemicals depolarize neurons and muscle cells.
   (d) Excitable tissues propagate action potentials along the membranes.
   (e) Schwann cells wrap their membranes many times round an axon's to make myelin sheath segments for faster signalling.
   (f) Chemical stimuli are transduced into nerve impulses in chemoreceptors; mechanical stimuli in mechanoreceptors.
   (g) Gap junctions permit ions and excitation to spread from cell to cell, and unify and synchronise actions of many cells/cell assemblies.
   (h) In development, epithelial and mesenchymal cells interact in sequence to induce cell differentiations, e.g., in tooth and glands.
   (i) Cells attract and fuse with one another to form multinucleated cells, e.g., skeletal muscle and osteoclasts.
   (j) Chemotactic agents act on phagocytic cells to attract them to their targets.
   (k) Keratinocytes of the skin phagocytose melanin pigment offered to them in the processes of melanocytes.
   (l) Macrophages detect spent or abnormal red blood cells, and hold and engulf either the whole cell, or the part holding an unwanted body.
   

3 Molecules Wherever such actions are described, special molecules are acting, by binding to each other, changing their conformation, or some other means. Examples are:
.. (a) Spectrin/fodrin provides a subplasmalemmal skeleton attached to the cell membrane by ankyrin, and to actin of the cytoskeleton, to permit control of the membrane's shape and movement.
.. (b) Cell adhesion molecules (CAMs) allow cells to attach to only certain cell types or substrates.
.. (c) Integrins are cell-surface-membrane dimeric molecules (an alpha with a beta), by which cells choose to which extracellular matrix (ECM) components they wish to fasten, e.g., laminin.
.. (d) Connexins are proteins that combine as hexamers to form connexons - the gap-junction channels, allowing ions and small molecules to pass between cells. Connexins and the transports allowed vary among liver cells, neurons, etc.
.. (e) Occludins are responsible for the seal preventing passage of materials past inter-epithelial tight junctions.

B INTERNAL (CYTO-) MEMBRANE SYSTEMS

l Nuclear membrane is a doubled membrane with pores that separates off genetic material and influences its degree and kind of interaction with the cytoplasm. The membrane sometimes has deep infoldings. (The rare annulate lamellae are parallel membranes with pores, and closely resemble nuclear membranes, but are stacked in the cytoplasm.)

2 Granular endoplasmic reticulum (ergastoplasm) is a membrane system providing some communication with the nucleus via the latter's outer membrane. Both have ribosome particles studding their outer sufaces, i.e., those facing outwards towards the cytoplasm. The membranes and ribosomes, in association with the nucleus, are concerned with protein synthesis. Some proteins pass into the sacs or cisternae, formed between the double membrane layers, for folding and processing. Membrane vesicles holding the protein bud off and travel along microtubules to the cis (receiving) face of the Golgi complex.

. Endosomes are the natural transport vesicles moving from the donor membrane compartment to the acceptor compartment. Endosomes require active mechanisms for: membrane budding, separation, transport, targetting & sorting, docking, and fusion. The donor and acceptor compartments may be the plasmalemma (via endocytotic vesicles), the Golgi complex, lysosomes, or the endoplasmic reticulum.
Some materials, e.g., receptors, are in a constant cycle between cell membrane, early endosomes, and late endosomes, with branch points for materials to come or go to the Golgi complex, lysosomes, or elsewhere in the cell.

[Note that microsomes are small bodies formed during separation by biochemical cell fractionation techniques: morphologically they are artefacts. They are derived from broken parts of the smooth or granular ER whose membranes 'heal' to form small vesicles, with or without ribosomes.]

3 Golgi apparatus serves as a hub for traffic out of, into, and around the cell. It is a complex transit region (there may be more than one in large cells) occupied by smooth-surfaced tubules, sacs and flatter chambers varying considerably in size. It concentrates, modifies and packages certain secretory products to await transport to the cell membrane for release, or application to some intracellular purpose. Vesicles depart from the trans or releasing face. Also, the Golgi is where glycoconjugates are finished by adding the remaining sugars (using glycosyltransferases), e.g., in cartilage cells and mucus-secreting goblet cells. Glyco

4 Many inclusions - structures not actively participating in the metabolism of the cell at the time of observation - are products bound in membranes by the Golgi apparatus, e.g., melanosomes, zymogen granules. Other inclusions, e.g., glycogen granules, form in the cytoplasm without any enclosing membrane.

5 Smooth endoplasmic reticulum resembles the granular form in sometimes having systems of parallel membranes following the curvature of nearby structures, but it usually exists in tubular and vesicular forms. Its functions are varied and include:

1. Acting as a source or reserve for membranes of new structures, e.g., Golgi body, GER; and cell membrane, as for the gastric oxyntic cell's canaliculi, and in the megakaryocyte's formation of platelets.
2. It is prominent in cells involved in cholesterol metabolism, e.g., liver cells and cells making steroid hormones. It seems to interact with lipid droplets, also present, in the synthesis of the steroids.
3. Smooth ER in liver cells is the site of enzymes detoxifying phenobarbital and other foreign chemical materials. It is also concerned there with the formation of plasma lipoprotein as a normal responsibility.
4. A smooth membrane system is found in muscle fibres as the sarcoplasmic reticulum. It synchronizes contractions and relaxations throughout the fibre, by moving calcium ions to and from the sarcoplasm.

6 Mitochondria
l These are complex bodies with a double membrane, the inner membrane extending inwards as sheets or tubules called cristae.
2 The inside of the mitochondrion is occupied by a matrix in which dense bodies may sometimes be found.
3 Enzymes of oxidation and energy-release, and for some syntheses, are present; some associated with the crista membranes or with the external/ outer limiting membrane, others and coenzymes are in the matrix.
4 Mitochondria of steroid cells are distinctive in having tubular cristae.
5 Mitochondria are able to reproduce themselves. Also, they contain circular DNA for 13 respiratory-chain proteins. One rare mitochondrial genetic disorder of this DNA causes, for example, a muscle disease with mitochondrial inclusion bodies.
6 Apoptosis involves mitochondria. Increased permeability of the outer mitochondrial membrane allows the release of inter-membrane factors, e.g., cytochrome c, that help start the caspase enzymatic cascade.

7 Lysosomes
l They are roughly spherical with a single enclosing membrane.
2 The storage/primary form is derived from the Golgi apparatus and contains hydrolytic enzymes,
3 whose access to other intracellular materials is controlled by the enclosing membrane and processes of membrane fusion. The stability of the membrane can be influenced by vitamin A and glucocorticoid hormones.
4 Lysosomes fuse with endosomes, phagosomes, surplus secretory granules or expended organelles, which they destroy.
Multivesicular bodies are endosomes on their way to meet lysosomes, but containing distinct vesicles within them, probably as a way to keep membrane separate and salvageable.
5 The form of lysosomes changes from round and fairly homogeneous to varied secondary kinds including myelin figures and residual bodies. Some residual bodies become yellow or brown lipofuscin/lipochrome granules.
6 In autophagy, the lysosome fuses with the autophagosome, consisting of a double membrane wrapped around the target organelles and cytoplasm to be broken down. . Autophagy PowerPoint
7 Lysosomal enzymes are also used in the turnover of extra- and intra-cellular materials. In lysosomal deficiency diseases, the inherited absence of an enzyme causes the massive accumulation of the material, e.g., glycogen, normally broken down. (Excess storage, hence "storage disease".)
8 A devastating storage disease is Hurler's, where a deficiency in lysosomal a -L-iduronidase causes intra- and extra-cellular excess accumulations of dermatan-sulphate and heparan-sulphate glycosaminoglycans. Aside from the dwarfism and mental retardation, the many cardiovascular defects bring about early death.

8 Peroxisomes/Microbodies are widespread, but particularly in hepatocytes and renal proximal tubule cells. They have a dense matrix enclosed by a single membrane, and hold enzymes involved in the beta oxidation of certain fatty acids. Catalase is a useful marker enzyme for peroxisomes. The congenital lack of peroxisomes causes a fatal syndrome with brain, liver and kidney dysfunctions.

C CYTOSKELETAL (FILAMENTO-TUBULAR) SYSTEM

The three components of the cytoskeleton - actin-myosin, microtubules, and intermediate filaments - are functionally linked as the dynamic organizer of the cellular domain, controlling cell shape, cell locomotion, where materials move in the cell, and hence cell polarity.

l Filaments

1. Several kinds of filaments, derived from unpolymerized precursors, attach to the plasmalemma, and to structures within the cell in order to change their form and position as the cell works.
2. Subdivision of filaments is by: (a) size - thick myosin (l5 nm diameter) and thin actin (6 nm) versus intermediate (l0 nm), roughly contractile versus noncontractile (also labile versus insoluble); and (b) by distribution - general versus tissue-specific.
3. Relatively tissue-specific intermediate filaments are: keratin in epithelia; desmin in muscle cells; neurofilaments in neurons; and glial filaments of acidic protein (GFAP) in certain glia. Immunostaining for these filament types gives some idea of the cellular origin of, and prognosis for, tumours.
4. Nestin is a protein closely associated with intermediate filaments in stem and progenitor cells of tissues generally, and serves as a stem-cell marker.
5. More generally distributed filaments are: (a) vimentin (intermediate) in connective tissue and other cells of mesenchymal origin, and in muscle, glia, and sometimes epithelia; (b) actin (thin); and (c) myosin, often not easily demonstrable as filaments.
6. In motile cells, e.g., white blood cells/leucocytes, the peripheral zone of cytoplasm clear of organelles and inclusions in phase-contrast microscopy, and used to extend pseudopodia, is the ectoplasm. This zone is now known as the actin cortex because of the actin filaments attached to the cell membrane for locomotion and changes in cell shape.
7. In absorptive and secretory epithelia, actin filaments are a major component of the apical terminal web inserting into junctional complexes and running up inside the microvilli. The actin is responsible for the movement involved in endocytosis and exocytosis.
8. Isoforms of actin (at least seven) have roles in striated muscle, smooth muscle, and non-muscle cells; and several proteins interact with these actins to achieve their controlled rapid reorganization for movement, e.g., profilin, a-actinin, filamin, severin, villin, etc.

2 Microtubules Each microtubule is built of 13 tubulin filaments. The dimers constructing the filaments confer a repeated polarisation along the tubule so that one end is 'plus', the other 'minus' - a difference that tells the attached microtubule motor proteins which end to head for.

1. Microtubules, not in a doublet or triplet formation, are supporting or cytoskeletal elements used, for instance: (a) to give some shape to platelets, (b) to cause and orient the elongation of cells, e.g., ameloblasts, (c) to allow neurons and glia to grow long processes and keep them patent; (d) microtubules are responsible for axoplasmic flow. (e) Microtubules direct certain materials to regions of the cell membrane, e.g., basolateral, creating cell polarity.
   Microtubules are dynamic structures in themselves and their construction and disassembly from tubulin dimers are under constant control.
2. Centrioles are cylinders open at one end with a wall composed of microtubules disposed longitudinally as nine triplets. The centrioles and additional microtubules direct and move the chromosomes along the mitotic spindle in cell division.
3. Cilia have, at their base, basal bodies, each identical with a centriole and often developing from one. Of the triplet, two microtubules extend up inside the cilium which thus has a core of nine peripheral doublets, plus an additional central pair. The doublets, by a sliding-tubule action using dynein arms, move the cilium to beat strongly in one direction by a bending-wave action.
4. Flagellum (tail) of the spermatozoon uses a cilium-like array of microtubules, aided by very thick fibres, for powerful swimming.
5. Intracellular movement of vesicles by microtubules is achieved by a repeating detachment and re-attachment of motor proteins - kinesin and/or cytoplasmic dynein - to the microtubule, energized by an ATPase. Two different motor proteins can achieve movement in opposing directions, as in axons, although the microtubules are polarised (+ & -) only one way.
6. Sensory cilia containing microtubules occur on cells in the special sense organs, e.g., olfactory, and play some role in sensory transduction.
   

D NUCLEAR-CYTOPLASMIC SYSTEM

2 Protein synthesis is the primary cell activity influenced by the nucleus. Proteins, as enzymes, transcription factors, receptors, and so on, are the key to the particular cell's character (Chapter 32).

3 Nuclear constituents are:

1. Chromatin - mainly DNA and dispersed at interphase (except for one female X chromosome); has a fine granular appearance in routine EM, but is fibrillar; some RNA is present.
2. Nucleolus/nucleoli - dense clumped granules; nucleic acid is mainly RNA, but some DNA is there. Peri-nucleolar/nucleolus-associated chromatin has a special relation to the nucleolus. A range in the number of nucleoli present usually exists, e.g., in the liver cells with one nucleus, l to 6, with 2 the modal value. Removal of DNA by DNase allows one to see nucleoli in the very dense nuclei of lymphocytes and some other cells.
3. Nuclear proteins - histones and non-histone proteins are associated with the DNA and RNA. A nucleosome comprises a length of DNA wrapped around a core of histones. The proteins mostly leave the nucleus during cell division.
4. Nuclear skeleton and karyoplasm/nuclear sap - A fibrous lamina is attached to the inner nuclear membrane, and supports the DNA periodically, creating intervening loops of DNA. The sap contains the more labile biochemical materials.
5. Histological methods for the nucleus:
   • Nuclear structure is seen in TEM.
   • Radioautography for rates of cell division uses tritium-labelled precursors of nucleotides, e.g., thymidine and cytidine. Bromodeoxyuridine (BrdU) is incorporated in place of thymidine, so that cells synthesizing DNA prior to division can be revealed by a labelled antibody to the BrdU.
   • Chromosomes and their banding patterns are studied in cytogenetics; see Chapter 30.
   • Fluorescent in-situ hybridisation/FISH uses 'coloured' nucleotide probes to identify a particular chromosome and, on it, the gene of clinical interest - normal, mutated, translocated, deleted, duplicated, truncated, etc.
   • Silver staining reveals the nucleolar organizer regions (NORs) as intranuclear black dots, because it marks the acidic proteins binding to the genes coding for ribosomal RNA located on certain chromosomes. An increase in the number of NORs so shown (AgNORs) correlates with dysplastic (abnormal) growth, and may indicate malignant tendencies in epithelial cells.

4 Protein synthesis (also Chapter 32)

1. Is controlled by messenger RNA, synthesized by transcription from the chromatin DNA, and leaving via the nuclear pores.
2. The nucleolus is a staging post for ribosomal RNA,
3. before it passes out to the cytoplasmic ribosomes, free or attached to membranes. Ribosomes are held in clusters by mRNA.
4. Transfer RNA brings to the ribosomes
5. activated amino acids for linking together according to the messenger RNA`s molecule (the translation step),
6. thus producing specific chains - a polypeptide or protein, with a sequence and length specified in the first instance by the transcribed nuclear DNA.

5
Cell fusion techniques, using the Sendai virus or chemicals, to produce hybrid cells have revealed that the cytoplasmic environment can alter the activity of the nucleus. Thus, a dense and inaccessible chromatin in a nucleus may be transformed by new cytoplasmic surroundings to one that is looser, paler and more reactive, as is seen in the small lymphocyte after antigenic stimulation. Cell fusion can also be between a nucleated cell and one made nucleus-free - a cytoplast.

E CYTOPLASM (cytosol/cell sap)

Structureless at the EM level, but of vital biochemical and physiological importance with its water, electrolytes, soluble proteins, amino acids, sugars, enzymes, etc. It is in continuous interaction with the filamentous and membrane-enclosed systems. Unwanted or defective proteins to be destroyed are conjugated with ubiquitin , which takes them to chemically structured concentrations - proteasomes - of proteolytic cytoplasmic enzymes Powerpoint.