Cell structure and the cell cycle

Major organelles and their principal functions

The three cytoskeletal filament systems

Membrane structure: key points

1. Membranes follow the fluid mosaic model: a phospholipid bilayer with integral and peripheral proteins that can diffuse sideways within it.

2. Fluidity rises with more unsaturated (cis double-bonded) fatty acids and with shorter chains. Cholesterol lowers fluidity at higher temperatures and prevents tight packing (keeping the membrane fluid) at lower temperatures, so it buffers fluidity.

3. Lateral diffusion is fast. Transverse movement from one leaflet to the other ('flip-flop') is very slow unless catalysed by flippases, floppases or scramblases.

4. The membrane is asymmetric. In the plasma membrane, phosphatidylserine sits in the inner (cytosolic) leaflet and glycolipids and glycoproteins face outward. Phosphatidylserine appearing on the outer leaflet is an 'eat-me' signal in apoptosis.

Other structures to know

1. Cilia and flagella (eukaryotic) have a 9+2 microtubule axoneme (nine outer doublets around a central pair) and are driven by axonemal dynein. Their basal body, like a centriole, has nine triplets and no central pair.

2. Features of plant cells that animal cells lack: a cellulose cell wall, plastids, a large central vacuole and plasmodesmata. Higher plants also lack centrioles, yet still form a mitotic spindle.

3. Features of animal cells that plant cells lack: centrioles within the centrosome, and lysosomes in the classical sense (the plant vacuole performs the lytic role).

Cyclin–CDK combinations and their cell cycle role (mammalian cells)

How CDK activity is controlled

1. Cyclin binding is required for any activity.

2. An activating phosphorylation (on Thr161 of CDK1) is added by CDK-activating kinase (CAK).

3. An inhibitory phosphorylation (on Tyr15 of CDK1) is added by Wee1 kinase and removed by Cdc25 phosphatase. Removal by Cdc25 is what triggers the burst of cyclin B–CDK1 activity at mitotic entry.

4. CDK inhibitor proteins (CKIs) block activity: INK4 proteins such as p16 inhibit CDK4/6, and Cip/Kip proteins such as p21 and p27 inhibit cyclin E–CDK2 and cyclin A–CDK2. The p53 protein induces p21.

5. Cyclins are destroyed by ubiquitin-mediated proteolysis: SCF targets G1/S cyclins and CKIs such as p27, and APC/C targets securin and the mitotic cyclins (A and B).

Cell cycle checkpoints, in order

1. G1/S checkpoint (the restriction point in mammalian cells, called 'Start' in yeast). It checks cell size, nutrients and growth factors, and DNA integrity before the cell commits to replication. It is controlled by the Rb–E2F switch. DNA damage activates p53, which blocks passage through p21.

2. Intra-S checkpoint: replication stress or damage during S phase slows origin firing.

3. G2/M checkpoint: it checks that replication is complete and the DNA undamaged before mitosis. DNA damage activates ATM (double-strand breaks) or ATR (single-stranded DNA and replication stress), which activate Chk2 or Chk1. These inhibit Cdc25, so cyclin B–CDK1 stays in its inhibited, Tyr15-phosphorylated state.

4. Spindle assembly checkpoint, at the metaphase-to-anaphase transition: an unattached kinetochore generates a signal (involving Mad2) that inhibits APC/C–Cdc20. Once every kinetochore is attached, APC/C–Cdc20 ubiquitinates securin and cyclin B for proteasomal degradation. Separase is then freed to cleave cohesin, the sister chromatids separate, and the fall in CDK1 activity allows exit from mitosis.

Stages of mitosis, in order

1. Prophase: chromatin condenses into chromosomes made of two sister chromatids, the centrosomes separate and the spindle begins to form, and the nucleolus disappears.

2. Prometaphase: the nuclear envelope breaks down (in animal cells; some fungi have a 'closed' mitosis) and spindle microtubules attach to the kinetochores.

3. Metaphase: the chromosomes line up at the metaphase plate.

4. Anaphase: cohesin is cleaved and sister chromatids separate. In anaphase A they move to the poles as kinetochore microtubules shorten; in anaphase B the poles move apart.

5. Telophase: the nuclear envelopes re-form and the chromosomes decondense.

6. Cytokinesis: in animal cells a contractile ring of actin and myosin II forms a cleavage furrow. In plant cells a phragmoplast guides Golgi-derived vesicles to build a cell plate.

Mitosis vs meiosis (meiosis shown for a diploid, 2n, parent)

Chromosome number and DNA content through meiosis (diploid 2n; C = haploid DNA content)

Meiosis I prophase substages, in order

1. Leptotene: chromosomes begin to condense.

2. Zygotene: homologous chromosomes begin pairing (synapsis) and the synaptonemal complex forms.

3. Pachytene: synapsis is complete and crossing over takes place between non-sister chromatids.

4. Diplotene: the synaptonemal complex dissolves and homologues stay joined at chiasmata, which become visible. Oocytes of many animals, including humans, arrest for a long period at this stage (the dictyotene stage).

5. Diakinesis: chromosomes condense further, the chiasmata move toward the chromosome ends, and the nuclear envelope breaks down in preparation for metaphase I.

Nondisjunction

1. Nondisjunction in meiosis I, when a pair of homologues fails to separate, makes all four gametes abnormal: two are n + 1 and two are n − 1 for that chromosome.

2. Nondisjunction in meiosis II, when sister chromatids fail to separate in one of the two cells, gives two normal gametes, one n + 1 and one n − 1.

3. Fertilisation of an n + 1 gamete by a normal gamete gives a trisomic zygote (2n + 1); an n − 1 gamete gives a monosomic zygote (2n − 1).

Secretory (co-translational) pathway, in order

1. The signal recognition particle (SRP) recognises a hydrophobic N-terminal signal sequence on the growing chain, pauses elongation, and docks the ribosome on the SRP receptor at the rough ER. This docking is GTP-dependent.

2. The chain enters the ER lumen through the Sec61 translocon, and signal peptidase usually cleaves the signal sequence. In membrane proteins, stop-transfer and internal signal-anchor sequences set the orientation and number of membrane crossings. Some proteins receive a GPI anchor in the ER lumen.

3. Correctly folded cargo leaves the ER in COPII-coated vesicles for the ERGIC and cis-Golgi.

4. Proteins are modified in sequence through the Golgi from cis to trans. ER-resident proteins that escape are retrieved in COPI-coated vesicles: soluble proteins by a C-terminal KDEL signal bound by the KDEL receptor, and membrane proteins by a C-terminal cytosolic KKXX (dilysine) signal bound directly by COPI.

5. In the cis-Golgi, lysosomal hydrolases are tagged with mannose-6-phosphate (M6P). In the trans-Golgi network, M6P receptors package them into clathrin-coated vesicles bound for late endosomes. There the acidic pH releases the enzymes, which reach lysosomes, and the receptors recycle. Failure of M6P tagging (I-cell disease) causes lysosomal enzymes to be secreted instead.

6. Other proteins leave the trans-Golgi network by constitutive secretion, the default route that needs no signal, or by regulated secretion from secretory granules in response to a signal.

Glycosylation and ER quality control

1. N-linked glycosylation: a pre-assembled oligosaccharide (${\text{Glc}}_3{\text{Man}}_9{\text{GlcNAc}}_2$) on dolichol phosphate is transferred in one piece by oligosaccharyltransferase to Asn in the sequence Asn-X-Ser/Thr, where X is any amino acid except Pro. This happens in the ER lumen, usually while the chain is still being made. The oligosaccharide is then trimmed and remodelled in the ER and Golgi.

2. O-linked glycosylation: sugars are attached to the hydroxyl of Ser or Thr, mainly in the Golgi.

3. Quality control: the lectin chaperones calnexin and calreticulin hold glycoproteins in the ER until they fold. Proteins that stay misfolded are moved back into the cytosol, ubiquitinated and degraded by the proteasome (ER-associated degradation, ERAD). Accumulated unfolded protein triggers the unfolded protein response (UPR).

Sorting signals and their destinations

Vesicle coats

Apoptosis vs necrosis vs autophagy

Apoptosis: extrinsic and intrinsic pathways

1. Caspases are cysteine proteases that cut after an aspartate residue. They are made as inactive procaspases. Initiator caspases (-8, -9, and -10 in humans) are activated by being brought together on a scaffold. Executioner caspases (-3, -6, -7) are activated when initiators cleave them.

2. Extrinsic (death-receptor) pathway: a ligand such as FasL or TNF binds its death receptor (Fas, TNFR1). The adaptor FADD then assembles the death-inducing signalling complex (DISC), which activates caspase-8.

3. Intrinsic (mitochondrial) pathway: stress or DNA damage activates BH3-only proteins (e.g., Bid, Bim, Bad, Puma, Noxa). These overcome the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1 and activate the effectors Bax and Bak, which permeabilise the outer mitochondrial membrane.

4. Cytochrome c released into the cytosol binds Apaf-1, which, with dATP or ATP, assembles into the apoptosome. The apoptosome activates caspase-9. Smac/DIABLO, released from mitochondria at the same time, neutralises the inhibitor-of-apoptosis proteins (IAPs, e.g., XIAP).

5. The two pathways are linked: caspase-8 cleaves Bid to tBid, which activates Bax and Bak and amplifies the extrinsic signal through the mitochondria.

6. Both pathways converge on executioner caspases, mainly caspase-3. These cleave structural and nuclear proteins, and cleave ICAD to free CAD for DNA fragmentation, producing the apoptotic morphology.

Autophagy: key points

1. Types: macroautophagy (a double-membrane autophagosome encloses the cargo), microautophagy (the lysosome membrane takes up cargo directly) and chaperone-mediated autophagy (proteins with a KFERQ-like motif are recognised by Hsc70 and moved across the lysosome membrane via LAMP-2A).

2. Regulation: when nutrients are plentiful, mTORC1 suppresses autophagy by inhibiting the ULK1 complex. Starvation, or the drug rapamycin, inhibits mTORC1, and AMPK activation at low energy promotes autophagy.

3. The Beclin-1–VPS34 (class III PI3-kinase) complex starts the autophagosome membrane.

4. LC3 (Atg8) is converted to lipid-linked LC3-II, which binds the autophagosome membrane and is a standard marker of autophagosomes.

5. The autophagosome fuses with a lysosome to form an autolysosome, where the cargo is degraded and its building blocks recycled.

Major ECM components

1. Collagen is the most abundant protein in mammals. It is a triple helix of three chains built from Gly-X-Y repeats, in which every third residue is glycine, X is often proline and Y often 4-hydroxyproline. Prolyl hydroxylation needs ascorbate (vitamin C), whose deficiency causes scurvy. Collagen is secreted as procollagen, its propeptides are cut off outside the cell, and lysyl oxidase cross-links it. Fibrillar collagens (e.g., type I in skin, bone and tendon; type II in cartilage) give tensile strength. Type IV collagen forms a sheet-like network in the basal lamina instead of fibrils.

2. Elastin forms a cross-linked network (via desmosine and isodesmosine, made from lysine residues) that gives elastic recoil. It is laid down on fibrillin microfibrils.

3. Glycosaminoglycans (GAGs) and proteoglycans are negatively charged polysaccharides that bind water and resist compression. In proteoglycans, sulfated GAGs such as heparan sulfate and chondroitin sulfate are attached to a core protein. Hyaluronan is the exception: it is not sulfated, is not attached to a core protein, and is made at the plasma membrane rather than in the Golgi.

4. Fibronectin is a dimeric glycoprotein that links cells to the matrix. Its RGD (Arg-Gly-Asp) sequence binds integrins.

5. Laminin is a cross-shaped αβγ heterotrimer and a major component of the basal lamina. It links cells to the type IV collagen network.

6. In plants the extracellular matrix is the cell wall: cellulose microfibrils embedded in hemicellulose and pectin.

Cell adhesion molecule classes

Cell junctions