Cells and Organelles Flashcards
What is the approximate resolving power of the light microscope?
0.5 microns
What is the method to prepare a histological slide for examination by light microscope?
Fix the tissue in Formalin to preserve the structure. (37% formaldehyde) - this crosslinks the proteins in the cell so they don’t break down
Embed the tissue on parafin wax
Section the tissue with a microtome to be placed on slides
stain tissue
What are the two dyes used for histological sections?
Hematoxylin and eosin
What are the properties of hematoxylin?
It is a cationic dye that resembles a basic dye. Reacts with anionic groups in cells and tissues. Stains nucleic acids very well, sulfate groups of glucosaminoglycans. Will stain rough ER because of mRNA. It is blue-purple. Substances stained by hematoxylin are described as basophilic
What are the properties of eosin?
Anionic dye, acidic dye, reacts to cations. Will stain cytoplasmic filaments and the cytoplasm in general, especially mitochondria. Also extracellular collagen type 1. Stains these pink-red. Eosinophilic = acidophilic
How might a physician rapidly look at a biopsy?
Sometimes it takes multiple days to properly process tissue, so they can rapidly freeze it in nitrogen and obtain the sectioning from a cryostat machine. This can be done in less than 10 min
How is a TEM sample prepared?
1 nm resolution
Fixed with glutaraldehyde to cross-link proteins in the cell
Tissue is stained with heavy metal. i.e. Osmium tetroxide. Imparts electron density to the cell and tissue structures
Placed in a resin such as epoxy (small bullets) for sectioning.
50 nm to 1 micron sections are cut using diamond knives
Polyclonal vs monoclonal antibodies
Polyclonal - many different antibodies which stick to the antigen of interest
Monoclonal - One specific antibody that will do this, produced by a single cell line
Why might indirect be preferred to direct immunofluorescence?
more than one tagged secondary antibody can bind to the primary antibody. Thus, the signal can be increased. However, you can get alot of background noise
What is a clinical application of monoclonal antibodies?
Can be used to treat certain carcinomas by binding antigens, such as the epidermal growth factor receptor (EGFR)
What is the structure of the nucleus membrane?
double lipid bilayer. nucleus is the site for RNA synthesis
what is the trilaminar membrane?
plasma membrane, plasmalemma, unit membrane
How does cholesterol align in the membrane? What is the function?
Hydroxy groups fit with the polar phospholipid head group. At low concentrations, it keeps the membrane from crystallizing. At high concentrations, it decreases membrane fluidity by stopping movement of integral membrane proteins
what are integral proteins?
Proteins partially or fully embedded in bilayer. Transmembrane will go all the way through
what is a lipid raft?
microdomains of self-associating membrane molecules. Outer leaflets = glycosphingolipids, which makes thicker plasma membrane in raft area. Lipid rafts are also enriched with cholesterol to hold them together. they are SIGNALLING platforms
what is the most common pump in the body?
Na+ / K+ ATPase - uses 1/5 of body’s energy
what is a channel protein?
allow for passive diffusion of small ions and molecules in either direction. I.e. Gap junctions
what is a structural protein?
form cell-cell anchoring or adhesion junctions
what is the glycocalyx?
The carbohydrate coat on the extracellular surface of cells composed of glycoproteins and glycoplipids. Can have enzymatic reactions in small intestine and kidney
how does freeze-fracturing work?
used to visualize the arrangement of intergral membrane proteins. freeze tissue specimens, chop with a knife blade, path of least resistance is usually between the phospholipid bilayer of cell membranes. metal replica of sample is prepared. Proteins appear as bumps on the image by TEM. P face is generally much more filled with proteins because it is stabilized by cytoskeleton
In freeze-fracturing, which face leaves pits and which face leaves bumps?
Pits - E face, from removed integral proteins that stay with P face
Bumps - P face (protoplasm), from intact integral proteins stabilized by cytoskeleton
what is an example of a binucleated cell?
liver hepatocytes
what is chromatin?
a complex of DNA, histones, and other nuclear proteins
what is the smallest unit of chromatin?
Nucleosome. Often called “beads on a string”. It is transcriptionally active
what is the densest form of chromatin?
Mitotic chromosomes
what is euchromatin vs heterochromatin?
Euchromatin is stretched out and transcriptionally active. This predominates in metabolically active cells. Electron lucent zones, and is less stained by hematoxylin.
Heterochromatin - transcriptionally inactive DNA, electron dense zones, more stained by hematoxylin “basophilic clumps”.
what are the three locations for heterochromatin?
- Marginal heterochromatin - located on perimeter of nucleus
- Karyosomes - clumps of heterochromatin throughout nucleus
- In the nucleolus
What is the function of the nucleolus?
site of ribosomal RNA transcription and processing, site of initially ribosomal assembly.
Where does the nucleolus arise?
It arises in specific chromosomal regions called nucleolar organizing regions. Remember that it has NO MEMBRANE but contains rRNA for initial assembly.
What are the three morphologically distinct regions in the nucleolus?
- Fibrillar centers - electron lucent zones filled with transcription factors and RNA polymerase 1 to transcribe the genes, at low concentration
- Fibrillar material - very electron dense regions containing ribosomal genes that are actively undergoing transcription, as well as a large amount of rRNA.
- Granular material - little dots on TEM of electron dense and electron lucent, representing immature ribosomes (pre-ribosomal particles).
What are nuclear lamins?
specialized intermediate filaments that make up the nuclear lamina. This is attached to inner nucleaer membrane and functions in nuclear organization, cell regulation, etc
What is the outer nuclear membrane continuous with?
the membrane of the rough ER. this means the perinuclear space is continuous with the lumen
what is the function of a nuclear pore?
Facilitates bidirectional movement of small molecules in and out of the nucleus. Proteins moving in must be tagged with nuclear localization signal, proteins moving out must be tagged with nuclear export sequence. Generally, mRNA and ribosomes go out, and histones and proteins move in.
how can a nuclear pore be identified?
The two bilayers of the nuclear envelope will meet by TEM. This is most easily seen when there is a break in the marginal heterochromatin
how can smooth ER be identified?
by TEM, smooth-surfaced circular, oblong, or irregularly shaped structures
where are three common places to find smooth ER?
- Steroid-secreting cells (testes, ovary, adrenal glands) - involved in steroid metabolism
- Hepatocytes - can convert lipid-soluble compounds to water-soluble for excretion in urine. Also involved in glycogen metabolism
- Skeletal and cardiac muscle - referred to as sarcoplasmic reticulum. It is a reservoir for calcium
What is the lumen of the rough ER called?
Cisternae. The folds of the rER are generally parallel
Where can ribosomes be found on the rough ER?
Always on the CYTOPLASMIC side
When might a cytoplasm be basophilic?
When it is mostly filled with rough ER, leaving mostly proteins and nucleic acids in the cytoplasm. This happens in cells that primarily produce proteins (i.e. plasma cells)
Where are proteins synthesized in the rough ER generally headed?
to the Golgi apparatus. They must be tagged to get there. Otherwise, the proteins would be made by polysomes
How does the cell know to take a protein to the rough ER?
There is a special 15-60 amino acid sequence that is bound by a signal recognition protein (SRP) which relocates the polysome to a docking protein on the rough ER. The docking protein (knocks off SRP + protein complex) aligns the ribosome with a translocator which dumps the protein into the rough ER after transcription is finished
What types of proteins are generally made by polysomes?
mitochondrial, peroxisomes, and nuclear
where are lysosomal hydrolases packaged?
the golgi
what are the faces of the golgi?
Cis - face closest to the rough ER
Trans - face farthest from rough ER
Medial - center, generally 2-4 stacks
TGN / CGN - Trans or cis golgi networks, another name for these faces.
Vesicles can be seen blebbing off both faces
what is the function of the Golgi?
Post-translational modifications of proteins, and packaging for cell delivery
What are the two models of Golgi protein movement? Which one is most likely correct?
Cisternae maturation model - proteins stay in one cisternae which matures from cis to trans
Vesicular transport model - proteins are transported from one face to another via vesicles.
-> most likely correct, since there is retrograde movement
What is COP-II
COP = COat Protein. Covers vesicles from rough ER bound for cis face of golgi
What marker in the trans-golgi network generally means a protein is bound for a lysosome or endosome?
Mannose-6-phosphate (M6P)
How does the golgi package a protein that will be an integral membrane protein?
It puts it in the membrane of a vesicle which will combine with the plasma membrane for exocytosis
What is good evidence for regulated secretory pathway proteins being released, rather than constitutive?
When you see many electron dense vesicles built up in the cytoplasm by TEM, it is a good sign of regulated secretion, since constitutive secretions just happen whenever and never really build up in the cytoplasm
What do lysosomes contain? what is the typical pH?
They contain acid hydrolases which are typically only active around pH 5. The membrane is always studded with proton pumps
Why don’t we ID primary lysosomes? How can you identify a secondary lysosome?
primary lysosomes look too much like peroxisomes.
Secondary lysosomes are vesicles with heterogeneous electron densities because it has begun to digest intracellular or extracellular materials
what type of a protein is an acid hydrolase? Why is this important for formation of lysosomes?
They are glycoproteins. Glycosylation patterns are modified in the golgi so that the M6P is exposed. It binds to a M6P receptor in the membrane of TGN. The receptor-protein complex and vesicle fuses with late endosome to become primary lysosome
How is a late endosome formed?
Formed by endocytosis. It becomes a late endosome whenever the pH has dropped to 5.0, and is ready to make a lysosome when acid hydrolases arrive. These may sometimes include particles that will initially be degraded when the hydrolases come
What is autophagy? How does it work?
A function of lysosome. When a non-functional organelle needs to be degraded, it is surrounded by smooth ER to make an autophagosome. This is fused with a lysosome for the organelle to be degraded, and then recycled or exocytosed.
What is phagocytosis?
When larger particles such as bacteria are engulfed by the cell, they can be degraded in lysosomes via destruction of phagosomes
What is the function of a lysosomal targeting signal?
it is a marker for cytosolic proteins to be brought to the lysosome for degradation
What is pinocytosis and why does it happen?
Also called fluid-phase endocytosis, this is the non-specific uptake of extracellular fluids and molecules that happens in nearly all cells. Happens nearly continuously, and replaces material lost by constitutive exocytosis
What are the functions of clathrin and adaptin?
They function in receptor-mediated endocytosis. Adaptin binds the integral membrane proteins which will bind target ligands. Clathrin binds to adaptin and forms a cage, which forms a “clathrin pit” or slight invagination on the membrane. Clathrin will ultimately encase the endocytosed vesicle
What are the fates of an internalized ligand-receptor complex brought in by a clathrin-dependent mechanism?
receptor is saved and ligand is degraded, receptor and ligand are degraded, receptor and ligand are saved, receptor and ligand are transported through the cell (i.e. epithelial cells moving IgA)
Where are peroxisomes most abundant?
Liver and kidney cells
What are the proteins functioning in peroxisomes? Where are they synthesized?
Catalase and peroxidases. Catalase is just meant to break down H2O2. They oxidize organic compounds such as fatty acids. Site of beta-oxidation of fatty acids too long-chain to be processed in mitochondria
What are the two types of cristae and where are they found?
Cristae are the structures created by folding up the mitochondrial inner membrane. Shelf-like cristae are the most common, but tubulovesicular cristae can be seen in steroid-secreting cells
What protein modulates the permeability of the mitochondrial outer membrane?
Porin
Where are cristae found to be characteristically thin and sparse?
Liver cells (hepatocytes)
What are the granules found in the mitochondrial matrix (inside inner membrane) for?
binding sites for calcium, with function unclear
What does the cristae containing in steroid secreting cells?
Enzymes for steroidogenesis
Where are proteins for the mitochondria produced?
Generally, on polysomes. They are sent to the mitochondria via a 5’ signalling peptide. However, some proteins are synthesized in the mitochondria
What are microfilaments composed of?
the protein actin. Smallest cytoskeletal protein and thus hardest to see
What are examples of class I and class II intermediate filaments?
Keratins. Also called tonofilaments or tonofibrils. I.e. acidic or basic cytokeratins found in epithelial cells
What are examples of class III intermediate filaments?
Vimentin / Vimentin-Like
Vimentin in fibroblasts and epithelial cells
Desmin in muscle cells
GFAP (glial fibrillary acidic protein) in astrocytes and Muller glia
What are Class IV intermediate filaments and example?
Neurofilaments
Nestin in neural stem cells
What are Class V intermediate filaments and example?
Lamins
Lamin A in the nuclear lamina of all nucleated cells
What are Class VI intermediate filaments and example?
Beaded filaments
Filesin in the lens of the eye
Where are microtubules held together in the cell? Roughly how thick are they?
They are 25 nm, the same size as a ribosome. They are held together and MTOCs called centrosomes, which have the same structure as basal bodies holding on cilia. This is near the nucleus
What makes up microtubules?
Dimers of the protein tubulin
What makes up the centrosome?
Centrioles plus pericentriolar material. Centrioles are 9 sets of three microtubules
Where are glycogen inclusions commonly found and what do they look like by TEM?
They look like clumps of polysomes, commonly found in hepatocytes and cardiac muscle cells
What is the storage form of lipids droplets and where are they often found?
Often found in steroid-secreting cells, they are stored as triglycerides
What do pigment inclusions look like?
Often yellow-brown stains seen in long-lived cells, formed by melanin and lipofuscin. Lipofuscin is collections of lysosomes that have fats which cannot be broken down anymore. Common in liver and neurons
What are crystals?
An uncommon type of cell inclusion, sometimes found in leydig cells in the testes
What is prophase?
DNA has already been duplicated during the S phase. Chromatin condenses and become visible as sister chromatids. Centrioles replicate and move on opposite sides to form the mitotic spindle. Nucleolus is disassembled
What is prometaphase?
Begins when nuclear envelope is disrupted into vesicular fragments. Kinetochore fibers (microtubules) attach on either side to the centromeres
What is metaphase?
All the chromosomes are aligned at the center of the mitotic spindle. Chromosomes are held in position at the metaphase plate by kinetochore microtubules
What is anaphase?
Sister chromatids separate and a cleavage furrow begins to develop
What is telophase?
Marks the end of nuclear division, kinetochore fibers disassociate and nuclear membranes reform. Chromosomes begin to decondense and nucleoli reform
What is cytokinesis?
Full division of cytoplasm
What characterizes necrosis?
Pathological or passive cell death. There is plasma membrane damage, swelling, and lysis. Associated with extensive tissue damage and inflammatory response
What is apoptosis?
Triggered by caspase cascade, the nucleus breaks down and the DNA fragments into chromatin bodies. The cell is either phagocytosed or breaks apart into several vesicles called apoptotic bodies which are phagocytosed (a result of MEMBRANE BLEBBING)
What does an apoptotic cell look like?
Dense basophilic nuclear chromatin fragments, with dark eosinophilic cytoplasm. Round or oval.
How are lamins regulated in mitosis?
Lamins (A (C), B) form the nuclear lamina. When phosphorylated, they become soluble and trigger the disassembly of the nuclear lamina. When dephosphorylated, they reform the nuclear lamina following completion of cell division
