Cell Biology - Exam III

COPI-coated vesicles

Retrograde vesicles; send materials “backward” compared to normal secretion order (i.e. from the Golgi back to the ER)

COPII-coated vesicles

Anterograde vesicles; send materials “forward” compared to normal secretion order (i.e. from ER to the Golgi)

• required for formation of vesicles budding from ER and moving to Golgi

Clathrin-coated vesicles

Endosomes that transport materials between the plasma membrane and Golgi

Sar1-GTPase

Initiates the formation of COPII-coated vesicles by…

1. Initiating membrane bending

2. Recruiting adapter molecules and COPII proteins

Arrival of vesicles at target membrane

Occurs via…

1. Tethering (or docking) at membrane

2. Fusion with membrane

Rab GTPase

Tethers vesicles to target membrane by Rab protein binding to Rab effector on target membrane

Trans-SNARE complex

Facilitates fusion of vesicles with membrane by forcing water molecules out of the “gap” between membranes

• t-SNARE on target

• v-SNARE on transporting vesicle

Functions of the Golgi apparatus

1. Glycosylation of proteins

   1. modifies N-linked oligosaccharides already added to proteins in ER

   2. Adds O-linked glycosylation to proteins on serine and threonines

2. Synthesis of proteoglycans

3. Sorting of proteins for return to the ER (have KDEL or KKXX sequence)

Lysosomes

“Intracellular stomachs” that…

• contain acid hydrolases (enzymes)

• have a pH of 4.6-5

• break down food, materials for recycling, contents of phagosomes, etc.

Pathways to degradation in lysosomes

Requires fusion of lysosomes with the following:

1. Endocytosis: endosome forms around small extracellular material

2. Phagocytosis: phagosome forms around large extracellular material

3. Autophagy: autophagosome forms around intracellular material

Phagocytosis pathway

1. Phagosome encloses particle

2. Phagosome merges with lysosome (containing digestive enzymes) to produce a phagolysosome in which contents are degraded, recycled, or exocytosed

Mannose 6-phosphate (M6P)

Targets lysosomal enzymes to the lysosome

Receptor-mediated endocytosis

Cell signaling receptors (integral membrane proteins) can be internalized for processing by early endosome, which removes the ligand and returns the receptor:

1. Ligand-bound receptors on cell surface accumulate in a coated pit

2. Clathrin-coated vesicles bud off from the plasma membrane surface

3. Clathrin coat is disassembled

4. Vesicles fuse with early endosome (recycling-sorting center)

5. Ligands are removed and degraded in the lysosome

6. Receptors are sent back to the plasma membrane

Dynamin

“Pinches off” calthrin-coated vesicles at the plasma membrane

Intermediate filaments

Maintain mechanical strength of only eukaryotic cells; include:

• Neurofilaments (neuronal cells)

• Keratins (epithelial cells)

• Nuclear lamins (all eukaryotic cells)

Structure of intermediate filaments

Polar monomers assemble into nonpolar dimers, which then assemble into 8 tetramers twisted in a rope-like filament

• assembly is controlled by phosphorylation and dephosphorylation

• they are strong and deformable (bend, but to do not break)

Microtubules

Provide organization for the cell via…

1. movement of chromosomes during cell division

2. directional intracellular vesicular transport

3. maintaining organelle orientation

Structure of microtubules

• Stiffest filament in animal cells

• Assemble into networks (normal cells) and spindles (mitotic cells)

• Held together by weak, non-covalent, and hydrophobic interactions for quick assembly and disassembly

• Comoposed of 13 parallel α and β-tubulin (which function as a GTPase) protofilaments, forming a hollow polar tube

Microtubule assembly and disassembly

Assembly is made up of two steps:

1. Nucleation (slow, rate-limiting step)

2. Elongation (rapid); occurs at the + end of microtubules

Rescue: MTs are stable and growing under high GTP levels, forming a GTP cap

Catastrophe: MTs are destabilized and disassembling under low GTP levels

Centrosome

• Composed of 2 centrioles

• Functions as a MTOC via α and β-tubulin heterodimer linkage with γ-tubulin

• Link to the - end of microtubules

Colecemid

Chemotherapy drug that disassembles microtubules via binding to tubulin and inhibiting formation; microtubules can still regrow after disassembly

Microtubule-associated proteins (MAPs)

Increase the stability of microtubules by linking them together

• e.g. Tau binds to microtubules when not phosphorylated; hyperphosphorylated Tau forms tangled clumps within cells

Kinesin

+ end-directed, ATP dependent MT motor protein; contains…

• a head that “walks” along the MT via ATP phosphorylation

• a coiled stalk

• a tail that binds to “cargo”

Dynein

- end directed, ATP-dependent motor protein

Myosin

ATP-dependent, actin-specific motor protein

Secondary cilia

Motile, 9+2 cilia containing A and B tubules, a central pair of MTs, a radial spoke, an inner dynein arm, and an outer dynein arm

Primary cilia

Non-motile, 9+0 cilia containing only A and B tubules

Actin

Mediates and maintains cell shapes; participates in…

• muscle contraction

• changes in cell shape

• cell-cell and cell-matrix junctional complexes

• cell division and cytokinesis

α-actin is found in muscle cell types while β and γ-actin are found in all cells

Actin monomers

G-actin; can join when bound to ATP (even though it can also bind to ADP)

Actin filaments

F-actin; 2 parallel protofilaments of G-actin form a right-handed helix; monomers are added at the + end

F-actin filaments remodeling

• “Seed” is added to the plus end in order for nucleation to start

• Monomers are added from the + end and lost from the - end

• treadmilling: constant state of equal adding and losing actin monomers

Nucleating proteins

Provide a template for actin monomers to add to at the + end (e.g. Arp2/3 complex)

Monomer sequestering proteins

Bind to actin-ATP monomers and prevent them from polymerizing (e.g. thymosin b₄)

End-blocking (capping) proteins

Regulate the length of actin filaments (e.g. capZ or tropomodulin)

Monomer-polymerizing proteins

Promote the growth of actin filaments (e.g. profilin)

Actin filament depolymerizing proteins

Bind actin-ADP subunits for rapid turnover of actin filaments (e.g. cofilin)

Cross-linking proteins

Alter the three-dimensional organization of actin filaments (e.g. filamin, villin, fimbrin)

Filament-severing proteins

Shorten filaments and decrease cytoplasmic viscosity (e.g. gelsolin, cofilin)

Membrane-binding proteins

Link contractile proteins to plasma membrane (e.g. vinculin, spectrin)

Myosin

Motor protein molecules that interact with actin

• Head binds to actin

• Tail binds to other myosin molecules

• Heavy chains can assemble into bipolar myosin filaments

Actin and myosin in cellular contraction (non-muscle)

1. Binding of ATP causes release of myosin from actin

2. Hydrolysis of bound ATP causes weak binding of myosin head to actin

3. Release of Pi causes tighter attachment and the “power stroke”

4. Release of ADP causes change in conformation for strong attachment (rigor state)

Actin and myosin in cellular migration

1. Portrustion of the leading edge (lamellipodium) is driven by actin polymerization

   1. Stimulus at cell surface activates WASP and Arp2/3

   2. Arp2/3 initiates nucleation in both main and side branches (in order)

2. Cell forms new attachments with substratum under lamellipodium

   1. Integrins: mediate connection with extracellular matrix proteins via clustering to form focal adhesions

   2. Focal adhesions anchor actomyosin stress fibers

3. The cell is pulled forward via actomyosin-mediated contraction

   1. Myosin II accumulates at rear of lamellipodia

   2. Actomyosin interactions generate contractile forces to “pull” cell behind leading edge

4. Adhesions toward the rear of the cell disassemble

Sarcomere

Assembly of actin and myosin into mobile bands that control muscle contraction; during muscle contraction…

1. M line (center) remains

2. H zone (center) shortens

3. A band (length of myosin) remains

4. I bands (ends) shorten

5. Z lines (ends) move closer together

Titin

Connects Z line to M line in sarcomeres, limiting the range of motion and contributing to passive stiffness of the muscle

Actin and myosin in cellular contraction (muscle cells)

1. Myosin ATPase synthesizes ATP that places the myosin head into the “cocked position”, initiating contact with actin

2. Myosin crossbridge swivel and pull actin toward M line (power stroke)

3. Myosin releases actin and resets to bind to another (doesn’t occur in unison)

4. Steps 2 and 3 are repeated multiple times

Calcium in muscle contraction

1. Ca²⁺ is released from the ER

2. Ca²⁺ binds to troponin, activating it

3. Troponin removes tropomyosin from myosin-binding sites on actin

4. Myosin binds tightly to and moves actin, which repeats as long as sites are uncovers and ATP is available

Gregor Mendel

Discovered that genes are a unit of inheritance; his postulates say…

1. Characteristics of organisms are governed by units of inheritance called genes

2. A reproductive cell (gamete) contains one gene for each trait while a somatic cell contains two

3. The pairs of genes are separated during gamete formation (law of segregation)

4. Genes controlling different traits segregate independently of each other (law of independent assortment)

Walther Flemming

Discovered chromosomes, but didn’t yet make the link between chromosomes and inheritance

Walter Sutton

Suggested that chromosomes follow Mendel’s laws and therefore carry genetic material; chromosomes are present as homologous pairs and form a bivalent during meiosis

Erwin Chargaff

Found that ratios of bases in nucleic acid were different

Pheobus Levine

Discovered the structure of nucleosides and the “tetranucleotide theory” which said that DNA is a repeating series of tetranucleotides

Hershey and Chase

Discovered that DNA is responsible for passing on genetic material

Watson, Crick, and Franklin

X-ray imaging proved the structure of DNA; it is

• composed of two antiparallel chains of complimentary nucleotides

• protected by a phosphate backbone on the outside

• a right-handed double helix

• polar (has a 3’ and 5’ end)

Highly repeated DNA sequences

1-10% of total DNA that has no known function

• Satellite DNA: >100 bp in a cluster; located within centromeres and used for species ID

• Minisatellite DNA: 12-100 bp repeats; often found near telomeres and used for DNA fingerprinting

• Microsatellite DNA: 1-5 bp repeats; used to analyze the relationships between different ethnic populations

Moderately repeated DNA sequences

DNA sequences repeated a varied amount of times; can be…

• Coding (e.g. genes for ribosomal RNA and chromosomal proteins such as histones)

• Non-coding (most of these sequences; can be…)

  • Short interspersed elements (SINEs)

  • Long interspersed elements (LINEs)

Single copy (non-repeated) DNA sequences

Organized into genes that code for proteins; accounts for less than 1.5% of the human genome

Nucleosomes

200 nt DNA unit composed of DNA and histones; each includes a core particle of supercoiled DNA that is wrapped around the histone core complex

Histone core complex

Consists of two copies each of H2A, H2B, H3, and H4, forming an octamer with H1 serving as a linker

Centrosome

Centrioles are replicated, functioning as an MTOC; formed during S phase of the cell cycle

Cohesin

Protein that holds sister chromatids together at the centromere

Kinetochore

Protein complex that assembles at the centromere to connect to chromosomes via microtubules

Prophase

1. Genetic material condenses into chromosomes (two chromatids attached at a centromere)

2. Cytoskeleton is disassembled and mitotic spindle is assembled

3. Golgi complex and ER fragment; nuclear envelope disperses

Prometaphase

1. Chromosomal microtubules attach to kinetochores of chromosomes

   1. MTs grow and shrink, as if “looking” for chromosome

   2. MT is stabilized once it makes contact with a kinetochore

   3. One MT from each spindle captures each chromosome

2. Chromosomes are moved near the spindle equator

Metaphase

1. Chromosomes are aligned along the metaphase plate, attached by chromosomal MTs to both poles

   1. MTs are undergoing treadmilling

2. Microtubules are remodeled

   1. Tubulin label added at the + end (kinetochore) of chromosomal MTs

Mad 2

Checkpoint protein that attaches to unpaired chromosomes in order to prevent initiation of anaphase

Anaphase

1. Centromeres split and chromatids separate

   1. Separase cleaves cohesin, allowing separation

2. Chromosomes move to opposite spindle poles (anaphase A)

   1. Astral microtubules: organize and move the centrosome

   2. Kinetochore microtubules: connect to kinetochores of sister chromatids

   3. Overlap microtubules: meet in the center of the cell and maintain structure

3. Spindle poles move farther apart (anaphase B)

Telophase

1. Chromosomes cluster at opposite spindle poles

2. Nuclear envelope assembles around chromosome clusters

3. Golgi complex and ER reforms

Cytokinesis

The cell membrane cleaves into two cells via a cleavage furrow, formed by actin and myosin interactions

Meiosis I

• Bivalents (pair of duplicated chromosomes) separate into single duplicated chromosomes

• Crossing over occurs from prophase to anaphase

• Ends as 2x, n

Meiosis II

• DNA is not duplicated

• Chromosomes separate into sister chromatids

• Ends as n

Spermatogensis

1. Primary spermatocytes (diploid) undergo meiosis I

2. Secondary spermatocytes (diploid) undergo meiosis II

3. Four spermatids (haploid) are formed

4. Spermatids mature into sperm cells (haploid)

Oogenesis

1. Primary oocytes (diploid) undergo meiosis I

2. Secondary oocyte and polar body (diploid) undergo meiosis II

3. One ovum and three polar bodies (haploid) are formed

Chiasmata

“Links” formed between duplicated homologous chromosomes as a result of crossing over; each pair of duplicated homologous chromosomes is held together by at least one chiasma

Down’s Syndrome

Caused by nondisjunction leading to Trisomy 21