Histology Flashcards
histology
study of cells, tissue, and organs at the microscopic level
EM and LM Resolution
for structures 0.25 um to 1mm use light microscope, 0.2nm to .25um use electron microscope (transition and scanning)
Processing tissue
fix (crosslink protein and maintain tissue archetecture; dehydrate; embed into hard material that can be but in to slices; section (cut into slices); stain
stains
charge-based H & E staining or immunochemical (LM)
omosium (ultra-thin sections of detailed structure for transmission EM
gold/metal coating for scanning EM for 3D like image
confocal microscopy
flourescent tags imaged using confocal laser LM can be used to optically section and make digital 3D reconstructions
Membrane
made of mainly phosphatidylcholine phospolipid which is ampipathic: hydrophobic and hydrophilic parts
membranes in TEM
under transmission electron microscope, membranes are trilaminar in appearence even though they are bilayers
-outermost layer=darkly stained polar head groups
middle layer=lightly stained hydrophobic tails from both layers
inner layer-polar heads from inner leaflet that stain darkly
Fluid-Mosaic Membrane Model
-cell membrane is fluid and phospohlipids and certain proteins can move quite freely
functions of cell membrane
- maintain cell integrety
- cell-cell interactions and selective permiability
- initiate transduction of extracellular signals to intercellular signals
ways proteins interact w/membranes
- integral membrane proteins span bilayer
- some covalently link to a fatty acid tail
- some covalently-link to a specialised phospholipid that inserts into membrane
- peripheral proteins (close in proximity) associate w/ an integral membrane protein
G-protein coupled receptors that generate cAMP messanger
-extracellular ligand/signaling molecule can be neurotransmitters or hormones
-receptor=g protein coupled receptor
-transducer=g-protein
-amplifier=adeylate cyclase
messanger=cAMP, which activates protein kinase A
GPCR-InsP3
receptor=GPCR
transducer=g-protein
amplifier=phospholipase C, which turns a molecule into messengers=InsP3 and glycerol, which activate protein kinase C and Ca signaling
endoplasmic reticulum
- made up of interconnected tubules and flattened sheets of membrane interconnected at 3-way junctions to form a network
- site of biogenesis for golgi and peroxisomes
- can fuse, split apart, branch to form junctions, move to different sites by cytoskeleton
SER and RER functions
- SER-lipid synthesis and calcium storage, storage of detox enzymes
- RER-protein synthesis, post-translational modification (glycosylation, adding disulfide bonds and folding)
unfolded protein response
- misfolded protein builds up in the ER and causes ER stress
1. ER-associated degradation (ERAD) of misfolded protein (in lysosomes or the proteosome)
2. upregulation of ER folding machinery (chaperones and lipid synthesis)
3. last resort apoptosis
cystic fibrosis
- mutation sin cystic fibrosis transmembrane regulator gene–>misfolding of the CFTR protein, which gets degraded by ERAD
- drug-mediated suppression of UPR to increase delivery of the CFTR protein to the membrane
Golgi structure and activity
-cis face receives vesicles from ER and trans face releases mature vesicles
-cis face: mannose phosphorylation
medial face-N-acetylglcosamine is added
trans face: addition of sialic acid and galactose, phosphorylation
vesicular transport model
-vesicles move cargo through golgi by budding off individual cisterna nad moving to the next while the golgi itself remains static
cisternal maturation model
cisternae themselves move though the stack carrying cargo
- small vesicles bud off and travel back along the stack to the cis face to recycle golgi enzymes
- this model has more evidence from direct fluorescent tagging of proteins imaged using confocal microscope
vesicular transport
-coat proteins (COP1, COPII, Clathrin) form spherical cages that recruit cargo and drive vesicle formation (which requires energy provided by GTP-bound GTPases)
targeting and fusion of vesicles
- initiation and cargo selection involves recruitment and activation of GTPase to membrane to recruit coat proteins and cargo
- coat assembles and drives vesicle formation and buds from membrane
- vesicle is release (scission), maybe mediated by coat
- hydrolysis of GTP (loses stability) releases coat
- tethering proteins target vesicles to correct target
- SNARES on vesicle and target membrane drive fusion b/t bilayers
exocytosis and endocytosis
exocytosis adds membranous material to membrane, endocytosis balances this
types of endocytosis
- phagocytosis-large particles like bacteria ingested
- pinocytosis-fluid, uses small vesicles, main process of endocytosis
- receptor-mediated: cell signaling, mediated by clathrin coats
trafficking of lysosomal enzyme
- lysosomal enzyme is phosphorylated on mannose in cic-golgi to generate mannose-6-phosphate
- M6P binds to M6P receptor in the trans-golgi, which recruits coat protein clathrin
- vesicle containing enzyme-receptor complex buds from trans-golgi
- vesicle uncoats and is targeted and fuses to late endosome
- pH releases lysosomal enzyme from M6P receptor
- receptor recycled back to golgi, enzyme is dephosphorylated and traffics to lysosome (pH activates it along w/dephosphorylation)
receptor mediated endocytosis=trafficking from PM
ex: lysosomal enzyme
- secreted lysosomal enzyme binds to M6P on PM
- complex recruits clathrin and endocytotic vesicle forms
- vesicle buds into cytoplasm, then uncoats and fuses with an early endosome then late endosome
- pH releases lysosomal enzyme and dephosphorylates it
- M6P receptor recycles back to PM or golgi, active enzyme trafficked to lysosome
retromers
multi-protein coat complexes that help recycle cargo-laden vesicles from endosomes back to TGN
autophagy
digestion of cellular organelles to provide building blocks for function and survival
tay-sachs disease
-no enzyme to brake down a specific type of lipid, so lipids accumulate in lysosomes which form residual bodies, especially in nerve cells
xanthomas
deficiency in LDL due to bad trafficking of LDL receptor
- cells can’t catabolize cholesterol and it builds up in blood and deposits in skin
- also develops atheroschlerotic plaques
lipid traffic
- in vesicles (within the membranes of vesicles that fuse w/other membranes)
- in lipid transfer proteins that are soluble in aqueous situation, most often across short cytoplamic gaps
- LTPs have hydrophobic pocket/barrel
- ex: cholesterol loads into barrel, LTP dissociates from donor membrane, fuses to acceptor membrane, and unloads cholesterol
LTPs location of action
often do lipid transfer where ER is in close proximity to other structures (stable ER junctions like triad in mm)
-LTPs can bind donor and acceptor membranes simultaneously and swing between them
classes of lipids
glycerolipids-glycerol backbone, hydrophilic choline and two fatty acids
sphingolipids-sphingosine backbone, some have choline and some have a sugar (glycosphingolipids), and one fatty acid
sterols-cholesterol based
mitochondria structure
outer membrane, intermembrane space, then inner membrane/cristae and matrix inside inner membrane
DNA contains only 37 genes so proteins must be coded by nucleus and imported
mitochondrial import
- chaperones bind the polypeptide to be imported and transport it to mitochondrion
- mitochondrial targetting sequence directs it to Translocon of Outer Membrane (TOM) complex (transmembrane) that drives polypeptide to intermembrane space
- small Translocon of Inner Membrane (TIM) complex (peripheral membrane protein) binds and delivers to large TIM complex (transmembrane) that inserts it into matrix
- polypeptide is released and folds into its structure
peroxisomes
small, self-replicating, membrane bound organelles that do beta oxidation
proteins trafficked with 1. peroxisomal targeting signal sequence
2. transfer of proteins from ER by fusion w/ER vesicles to peroxisomal membrane
cytoskeleton
3D network of protein filaments that maintain cell morphology and move intercellular components and facillatate entire cell migration
actin cytoskeleton
- thinnest filaments-fiberous actin comprised two globular actin chains
- capping proteins regulate length and speed of growth of filaments, which can form bundles for different functions
- myosin motors can attach to initiate contraction or move cargo
- focal adhesions where actin binds to ECM across PM
microtubules
hollow tubes composed of tubulin heterodimers that can grown from + end by adding dimers
- origionate from centrosome from gamma tubulin ring complexes (-) end
- critical for movement of organelles and cellular projections like cilia and flagella
- centrioles within centrosomes nucleate microtubules and generate spindles
- kinesin motor proteins take cargo from (-) to (+) end
- dynein motor proteins move cargo from (+) to (-) end
intermediate filaments
- intermediate in diameter, formed by overlapping protein roads
- form structural scaffolds in cytoplasm and nucleus
euchromatin
heterochromatin
light patches, unwound DNA for transcribing
densely packed
nuclear pores
9-11nm, number correlates w/metabolic activity of cell
Ran GTPase cycle
in cytoplasm, cargo proteins interact w/importins and the complex moves through nuclear pore
- in nucleus, RanGTP binds complex and releases cargo, then leaves the nucleus w/importin
- in cytoplasm RanGAP converts RanGTP to Ran GDP, turning it off and releasing importin protein
- Ran GDP diffuses back through nuclear pore (small) and in converted to Ran GTP by Ran GEF
cell cycle checkpoints
- cell can only move forward when appropriate combo of cyclins and cyclin dependent kinases are present
- eg when DNA damage activates p53, which induces production of p21 which stops the cell from moving from G1 to S
1. if damage is not fixed when this happens, p53 can then direct apoptosis via intrinsic pathway
apoptosis
- extrinsic pathway: death ligands bind to death receptors and initiate signaling pathways that activate the executioner caspases
- intrinsic pathway-intercellular stresses like DNA damage initiate release of cytochrome C from mitochondria, which activates apoptosome, which activates executioner caspases
