Exam 1 Flashcards
(46 cards)
6 steps of preparing histological section
Steps of preparing histological section:
1. Tissue collection
-most common: needle biopsy or endoscopic biopsy
-less common: transvascular, direct excision, curettage
2. Fixation: preserve tissues by preventing degradation while maintaining nml tissue architecture.
-chemical fixatives: formaldehyde and glutaraldehyde
-dehydration: alcohol
-rapid freezing
3. Dehydration and clearing
-alcohol
4. Embedding and Sectioning
● Paraffin wax
-Standard method for preparing thin (5 μm-8 μm) sections for light microscopy
-Good resolution of cell structure and tissue architecture
-Slow (~ 24 hours)
●Acrylic resin
-Preparation of thin (1 μm) sections used in high resolution light microscopy
-Preparation of ultrathin (60 nm-80 nm) sections for EM
-Incompatible with most histological stains
-Slow (several days)
● Frozen sections
-Thick sections (12 μm-20 μm) and relatively low resolution
-Ideal for many histochemical and immunological stains
-Rapid (minutes - hours)
5. Mounting and staining
6. Viewing
Common stains
●Hematoxylin-Blue: basic dye that binds to acidic structures in cell; cartilage matrix
●Eosin-Pink: acidic dye; binds to basic regions of cytoplasm; collagen fibers
●Wright’s stain (methylene blue and eosin)- used for blood cells. Pink: eythrocytes, eosinophil granules. Blue: nuclei of white blood cells, cytoplasm of monocytes and lymphocytes
●PAS (periodic acid-Schiff) stain- stains carb/glycogen rich components of tissues and
cells a magenta color
ones we didnt talk about:
●Masson’s trichrome: dark blue – nuclei, red – muscle, keratin, cytoplasm, light blue – mucinogen, collagen
●Weigert’s elastic stain: blue – elastic fibers
●Silver stain: black - neurofilaments, reticular fibers
●Iron hematoxylin: black – striations of muscle, nuclei, erythrocytes
Different methods of viewing histological sections (include resolution!)
• Human eye - 0.1 - 0.2 mm
• Light microscope - 0.25 μm (could do RBC, staph bacterium)
• Transmission electron microscope - 0.2 nm (needed to see polio virus)
-TEM for looking at sections of structures
-Staining with heavy metal salts such as uranyl acetate and lead citrate provides contrast
for sections viewed in the EM
-provides contrast by blocking passage of electrons
• Scanning electron microscope - 10 nm (needed to see polio virus)
-SEM for looking at surfaces of structures
-SEM images are generated by analyzing the pattern of electrons reflected from a thin layer
of heavy metal (gold, palladium) deposited on the surface of the specimen
1. Autoradiography
• Incorporation of radioactive isotopes into macromolecules, which are then visualized
by use of an overlay of film emulsion
2. Enzyme histochemistry
• Cleavage of artificial substrate results in deposition of insoluble colored reaction
product at site of enzyme activity
3. Immunocytochemistry
• Binding of labelled antibodies enables visualization of macromolecules
Fluid mosaic model of the plasma membrane
●Lipid molecules are amphipathic (2 diff affinities); form bilayer in aqueous medium
●Cell membrane is fluid, allows lateral diffusion of lipid molecules and membrane proteins
●Membrane is impermeable to charged ions but permeable to small nonpolar and small polar molecules
●Membrane proteins perform functional roles such as transport, signaling, adhesion
Name 3 types of membrane proteins
phospholipids, cholesterol, glycolipids
Membrane proteins: phospholipids (how is it named, structure, “kink”)
-50% or more of total lipid in bilayer
-Amphipathic - have both hydrophobic and hydrophilic regions
-Named after amino-alcohol that forms head group e.g. phosphatidylcholine,
phosphatidylethanolamine
-Kink (one C=C double bond) in unsaturated fatty acid chain helps maintain membrane
Fluidity, doesn’t allow close packing
-Phospholipid bilayers are impermeable to charged molecules but permeable to small
uncharged molecules
**see image: Amino alcohol, connected to phosphate bridge=hydrophilic polar head
Glycerol backbone
Double unsaturated fatty acid chains (hydrophobic/nonpolar)
Membrane lipids: cholesterol (4 functions)
-Varies from cell to cell but can be almost as abundant as phospholipids
1. Provides mechanical strength, conversely, at high conc it resists close packing of f.a.’s
2. Acts as membrane fluidity buffer
3. Reduces permeability to small molecules
4. Contributes to formation of microdomains known as lipid rafts (important for cell
recognition)
Membrane proteins: glycolipids (where are they located? function? disorder?)
- 5-10% of total lipid
- Present only in outer leaflet
- contribute to glycocalyx (sugar coating over surface of most cells-produces a good stain)
- ex: tay-sachs disease- defect in ganglioside metabolism
Membrane proteins (function, 2 types)
- account for ~50% of mass of membrane
- Responsible for membrane function including:
- Transport of ions and molecules
- Cell-cell recognition and adhesion
- Signal transduction
- Two forms of membrane proteins:
- Integral membrane proteins
- Peripheral membrane proteins- glycocalyx
Glycocalyx (what is it? function?)
●Glycocalyx (sugar coating over surface of most cells-produces a good stain)
- Present only on extracellular surface of cell
- three functions:
1. Strong negative (polyanionic) charge on outer surface helps block infection
2. Protects cell from chemical or mechanical damage
3. Important for cell-cell recognition
Describe 3 types of endocytosis
- Pinocytosis (cell drinking)
- Bulk fluid uptake into clathrin coated endocytic vesicles
- Pinching off of vesicles driven by a collar of GTPase protein called dynamin.
- Vesicles usually fuse with lysosome but in capillary endothelial cells vesicles fuse with membrane opposite to their origin – transcytosis
- Helps meet general cell requirements for nutrients
- Important for maintaining membrane surface area, shape and size of cell
- Receptor-mediated Endocytosis
- take up specific macromolecules (ligand)
- specific example: cholesterol (LDL) uptake - LDL receptors on cell surface bind loose LDL
- clathrin coated pit forms into vesicle (clathrin coating with bound receptors inside)
- Becomes an early endosome, acidified interior uncouples LDL receptor to LDL.
- *Thus is also called CURL (compartment for uncoupling receptors and ligands)
- LDL receptors returned to surface, LDL sent to its destination
* *interesting side note: at this stage, if the specific macromolecule/ligand needed to be degraded, it would be sent to a late endosome which would fuse w acidic/lysosomal hydrolazes to become a lysosome and destroy contents - Phagocytosis (only phagocytic cells, for bacteria/cell fragments)
- Process of engulfing particulate matter
- Generally restricted to macrophages and neutrophils
- Triggered by binding to receptors in cell membrane
- Driven by rearrangement of actin cytoskeleton (clathrin independent)
- Large vesicle containing engulfed material called a phagosome
exocytosis (describe, 2 types)
Exocytosis (2 types):
●Fusion of membrane bound cytoplasmic vesicle with cell membrane
●Constitutive pathway important for replacing membranes and membrane proteins as well as secreting materials synthesized by cell
●Regulated pathway controlled by specific stimulus
●Fusogenic proteins that catalyze vesicle fusion with membrane vary depending on pathway
Types:
1. Constitutive exocytosis
-unregulated, ex: mucous granules
2. Regulated exocytosis
-waits for signal, such as hormone
Smooth ER (functions)
●Generally less abundant than RER
● Functions:
- Membrane synthesis
- Steroid hormone synthesis (Ie endocrine and reproductive systems)
- Detoxification of lipid soluble drugs, metabolic waste and ingested toxins by cytochrome
P450 system
- Ca2+ homeostasis e.g. sarcoplasmic reticulum
Rough ER (describe, function)
- Cytoplasmic surface studded with ribosomes
- Continuous with nuclear envelope
- Participates in synthesis of integral membrane and secreted proteins
- Initial site of posttranslational modification of proteins and lipids
Ribosomes (size, location, parts, function. What is polysome/polyribosome?)
- Free in cytosol or bound to RER
- 12 nm wide by 25 nm long
- Consist of small (40S) and large (60S) subunit
- Catalyze protein synthesis (translation)
- Polysomes (polyribosomes) reflect simultaneous translation of single mRNA by multiple
ribosomes
Golgi Apparatus (whats it for, functions, faces, other components)
• Default destination for proteins synthesized in rER
• Functions:
1. Further post-translational modification of proteins
2. Assembly of multisubunit proteins
3. Protein sorting and packaging into secretory vesicles
Consists of:
—-RER—–
Endoplasmic reticulum/Golgi intermediate compartment (ERGIC), now known as or perhaps also known as Vesicular-tubular clusters (VTC): outermost cisterna of cis face made of interconnected tubes/vesiles
Cis face (forming face): next to RER
Medial face (intermediate face): several cisternae lying bw cis/trans
Trans face (exit face): faces p.m., assoc w vacuoles and secretory granules
Trans Golgi network (TGN): last cisterna at trans face, separate from golgi stack. Sorts proteins for final destination
Lysosomes (size, where do they come from, what is a late endosome?, enzymes within lysosomes, disorders)
-cellular organelles that contain acid hydrolase enzymes that break down waste materials and cellular debris.
1. Small ( < 1 μm), irregularly shaped vesicles filled with acid hydrolases
2. Derived from trans Golgi network
- Membranes contain mannose-6-phosphate receptors that bind lysosomal enzymes
(my version: lysosomal proteins get processed thru golgi, mannose 6 phosphate is added, thus can be recognized by mannose 6 phosphate receptors and packaged into vesicles to go to endosomes)
3. Secondary lysosomes (or just lysosomes in general I think) result from fusion of late endosomes with lysosomal/acidic hydrolases and lysosomal membrane proteins coming from Golgi **see earlier note in endocytosis: endosome + hydrolases = lysosome
4. Enzymes (proteases, nucleases, glycosidases, aryly sulfatases, lipases, phospholipases, phosphatases)…. Hydrolytic ie low pH enzymes
heterophagy vs autophagy
Heterophagy: intracellular digestion of material taken into cell from outside
Autophagy: digestion of parts of the cell (ie damaged organelles)
lipofuscin
membrane-bound, electron dense granular material often containing lipid droplets. Represents residue of undigested material present in residual bodies. B/c residual material inc with age, it is called age pigment. Most common in non-dividing cells.
Peroxisomes (other name, size, contains which enzyme?, 2 functions, how do they replicate?):
- Also known as microbodies
- ~ 0.2 μm diameter
- Contain catalase (breaks down hydrogen peroxide) and other oxidative enzymes
- Function:
- Break down of long-chain fatty acids to produce Acetyl CoA and hydrogen peroxide
- Detoxification - Replicate by fission
- Enzymes synthesized in cytoplasm
Mitochondria (shape/size, function, matrix vs inner mito membrane, how do they replicate?)
- Rod-shaped, ~ 0.5μm diameter and up to 7 μm in length
- Produce ATP via oxidative phosphorylation
- Matrix space: (mito center)
- Enzymes of tricarboxylic acid (Krebs) cycle
- Matrix granules
- Circular DNA
- mRNA, ribosomes, tRNA - Inner mitochondrial membrane. High proton gradient (high proton (+) conc in i.m. space)
- ATP synthase protein complex (elementary particles visible on negatively stained cristae)
- Enzymes of electron transport chain
- Rich in cardiolipin (phospholipid with 4 f.a.’s, impermeable to electrons and protons) - Self replicating
**mitochondria inherited from mother. Individuals are mosaics of genetically distinct mitochondria. Manifestations of having dysfunctional mitochondria, ie “mitochondrial cytopathies”=
–increased lactic acid bc they use glycolysis/aneorobic (don’t have mitochondria for oxidative
process)
–terminally differentiated cells most severely affected, ie see them in cells that don’t replicate often ie muscle/neuro, not epithelial (cells with bad mito’s just die off and replaced w good ones)
Inclusions (define, examples)
Inclusions= accumulations of material this is not metabolically active, usually present in cytosol only temporarily
•Glycogen granules – energy storage
•Lipid droplets – triglyceride/chol storage
●centrosome
•Pigment granules
- Lipofuscin, melanin
•Crystalline inclusions
Nucleus (describe inner/outer membrane, two types of chromatin, nucleolus, define importins and exportins)
- Nuclear envelope
- Inner nuclear membrane supported by nuclear lamina composed of lamins A, B, C
- Outer nuclear membrane continuous with RER - Chromatin - complex of DNA and proteins
- Heterochromatin - condensed, inactive
- Euchromatin - dispersed, transcriptionally active - Nucleolus
- Site of ribosomal RNA synthesis and assembly of ribosome subunits
describe nuclear core complex
Nuclear pore complex:
- Nuclear pores facilitate passage of molecules between nucleus and cytoplasm
• Allows free passage of ions and molecules ≤ 60 kDa
• Facilitates bidirectional active (energy dependent) transport of larger molecules
• Nuclear localization signals (importins)
• Nuclear export signals (exportins)
