midsem (mod 1&2)

• Nucleus


  Large organelle, gene expression (transcription) determines the nature of the cell/organism, complex organization, the brain of the cell, highly dynamic

  
• Nuclear membrane


  The nucleus is surrounded by two membranes + nuclear lamina, inner nuclear membrane defines the nucleus, outer nuclear membrane is continuous with rough endoplasmic reticulum, separated by perinuclear space

  
• Nuclear lamina


  Meshwork of filaments located adjacent to the inside face of the inner nuclear membrane, made up of intermediate filaments (nuclear lamins), provides nucleus with structure

  
• Nucleolus/nucleoli


  A sub-organelle and clearly defined structure within the nucleoplasm, no membrane, site of ribosome biogenesis, formed around regions of DNA encoding ribosomal RNA, hotspot of transcriptional activity
• Nuclear bodies


  Membraneless nuclear sub-compartments, highly dynamic
• Chromatin


  DNA+histone complex: nucleosome, packaging of over 2m of DNA within nucleus, chromatin structure is dynamic (extended/condensed), chromatin structure determines gene expression

  
• Regulation of chromatin structure


  Histone tails can be targets of several post-transcriptional modifications, unacetylated = tightly coiled, transcriptionally inactive (HETEROCHROMATIN), acetylated = less condensed, transcriptionally active (EUCHROMATIN)

  
• Transcriptional machinery


  1. Histone acyltransferases bind to DNA transcriptional activation regions to recruit chromatin remodeling complexes to unwrap chromatin structure

  2. 2a. They also recruit a protein bridge (mediator) to help recruit transcription factors to a promoter sequence

  3. 2b. Mediator complex facilitates assembly of the preinitiation complex that includes leading an RNA polymerase (RNA pol II, exclusively for mRNA; pol I is for rRNA) on DNA

  4. After initiation, transcription is paused by an elongation factor complex to ensure RNA polymerase is properly loaded

  5. Elongation pause is relieved by phosphorylation and remodelling of the elongation factors by a cdk/cyclin pair

  
• Ribosome biogenesis


  Ribosomal RNA is first transcribed by RNA Pol I as a large transcript (pre-rRNA) that is then processed to 28S, 18S and 5.8S mature rRNA found in ribosomes, process similar to processing of pre-mRNA into mature mRNA, 5s transcribed in nucleoplasm by RNA Pol III and diffuses into nucleous, 60s and 40s ribosomal subunits undergo a quality control prior to export into cytoplasm through nuclear pores and mediated by nuclear export adaptors, final assembly into 80S, the functional translation machinery, occurs in cytoplasm
• Nuclear Pore Complex


  Spans both nuclear membranes, only gateway in or out of nucleus, allow passive diffusion by small molecules under 40 kDA, any bigger molecules needs to be guided through, pore is big enough to fit the larger of the ribosomal subunits (60S, ~25nm), gigantic ~125MDa in size, barrier and transport Nups (FG-Nups) in cytoplasmic filaments, central channel and nuclear basket facilitate high-specificity binding of transporters and their cargos

  
• Nuclear transport


  1. Localization signal on the cargo: import signal = nuclear localisation sequence (NLS), export signal = Nuclear export sequence (NES), cargo may have multiple localisation signals

  2. Delivery/Transporter System: recognise localisation signal on cargo to form a cargo complex, overcome size limit barrier of NPC to allow movement through the pore, transporters for nuclear import = Importins, transporters for nuclear export = Exportins, Importin/Exportin are receptors for FG-Nups

  3. Control system for delivery: utilises a GTPase switch, binding of cargo transporter (importin/exportin), key GTPase switch is a small G-protein called Ran-GTP/GDP, Ran-GTP and Ran GDP (23kDa) can randomly diffuse through nuclear pore

  
• Nuclear Import Mechanism


  1. 1a) Ran-GTP binds importins with high affinity

  2. 1b) This causes importins to release their cargo

  3. 2a) Ran-GTP/ Importin diffuse back into cytoplasm

  4. 2b) Cytoplasmic GAP (only found in the cytoplasm) activity converts Ran-GTP to Ran-GDP lowering affinity and release Importins

  5. Importins are recycled to transport more cargo

  6. Ran-GDP randomly diffuses back into nucleus to be converted into Ran GTP by nuclear GEFs (enriched in the nucleus as they are bound to the chromatin)
• Nuclear export mechanism


  Almost identical to nuclear import, except that Ran-GTP binding of exportins PROMOTES exporting, and releases cargo and exportin when converted to Ran-GDP by GEF

  
• Endoplasmic reticulum


  ER sheets and tubules: rough ER has ribosome on its surface, smooth ER has a highly branched, "tubular" morphology, the whole thing is one continuous network with common luminal space, continuous with the nucleus
• Dynamic ER membranes


  ER membrane movement extension and retraction coordinated with microtubules, ER linked to MRs and grow as MTs grow or, dragged along MTs through action of MR motors

  
• Shaping of ER membrane


  Phospholipid bilayers tend to be flat as curving membrane requires energy expenditure, "reticulons" - an ER membrane protein are responsible for membrane curvature, reticulons are inserted into the ER membrane in a wedge-like conformation to curve the phospholipid bilayer, several reticulons give the sharp curvature of tubes and at the edges of ER sheets
• ER membrane fusion by small G proteins


  3 way branching = fusion of an extending tube with side of another tubule, a small GTPase, Atlastin, is responsible for 3 way branched structure of ER tubules, Atlastin-GTP from opposite membranes form a dimer (dimerization), GTP hydrolysis somehow draws membranes together for fusion
• Endoplasmic reticulum function


  Rough ER: the ribosome on its surface bind to mRNA, translate them into the membrane and synthesise protein
• Getting a protein into the ER lumen


  ER targeting needs to happen at the same time as protein synthesis - "cotranslational translocation", there is an adaptor complex, the signal recognition particle SRP, there is an SRP receptor in the ER membrane, translation halted until the ribosome gets to ER translocon, docking of the SRP to its receptor opens up a channel allowing the translocation of the newly synthesized peptide, signal peptidase cleaves the signal sequence off the polypeptide, polypeptide folds within the lumen of the ER
• ER targeting and translocation: membrane proteins


  • Type I: initial steps are identical to translocation of secreted proteins, insertion into membrane requires a "stop-transfer anchor" (STA) signal
  • Type II: DO NOT have a cleavable N-terminal signal sequence, instead translation initially occurs in cytoplasm, however, an internal ER targeting sequence is then recognised by SRP and directed to ER translocon, this internal targeting signal also doubles as an anchor signal: a "signal anchor sequence" (SA), once SA sequence is embedded, it is moved laterally along the bilayer and ribosome continues cotranslation into ER lumen, reverse topology compared to Type I membrane proteins, cytosol first, lumen second, positive charge before the SA embedding
  • Type III: same topology as Type I but translocation mechanism is similar to Type II, does not have a cleavable N-term signal seq, uses a signal-anchor sequence but positioned very close to N-terminus, recognised by SRP, brought to translocon and anchored into membrane but in reverse orientation to Type II (hence reverse topology), positive charge after SA embedding
• Golgi Apparatus


  Two faces: Cis Golgi (convex) - the face that points to the ER, whenever the ER done synthesising a particular protein, the protein, covered by a vesicle that has specific cop II proteins that is exclusive to the cis golgi surface, Trans golgi (Concave) - after modifications occur inside of the Golgi to that protein, it is secreted and become a part of the membrane or turned into lysosomes

  
• Golgi machinery


  Intact microtubules is required to maintain the golgi's ribbon structure and perinuclear localization, during mitosis, the microtubules are reorganised into spindles for chromosome segregation, resulting in dispersion of the Golgi (into mini-stacks and individual cisternae)

  
• Golgi Transport: ER to the golgi


  Newly assembled proteins move from ER to cis-Golgi in transport vesicles that gets pinched off the ER, this forms a protein coat vesicle, coat needs to disassemble prior to membrane fusion at target organelle, COPII coated vesicles move from ER to golgi (anterograde transport), COPI vesicles move in reverse direction, golgi to ER (retrograde transport), sorting signal on cytoplasmic domain of membrane cargo proteins are recognised by coat proteins and selected for inclusion in budding vesicle, soluble cargo proteins (those that are not membrane proteins, not embedded) with luminal sorting signals require recognition by membrane cargo receptors for transport selection
• Golgi transport: retrograde retrieval


  Proteins may also be randomly captured by budding vesicles, thus need COP I, the sorting signal for ER resident proteins is the KDEL peptide sequence (for retrograde), slight difference in pH in ER vs Golgi determines binding of KDEL to a membrane receptor for retrieval
• Golgi function: Glycosylation in Golgi


  Distinct reactions in different Golgi compartments, because cis-, medial-, trans- compartment contain different sugar modifying enzymes, the process of modification is sequential, the product of one later is the substrate for the next, akin to an assembly line, "cisternal maturation": the glycoproteins do not move from one compartment to another, rather, the compartment itself matures, i.e, the cis face cisternae mature and move towards medial and then trans face, and replaced by newly formed cis-cisterna
• Golgi function: retrograde retrieval


  The enzymes of each compartment stays the same due to retrograde retrieval, i.e: cis-golgi enzymes retrieved from medial compartment by COPI vesicles moving in retrograde fashion, medial-golgi enzymes retrieved from trans compartment in same way
• Diseases associated with golgi
• cis-


  Compartment containing sugar modifying enzymes
• medial-


  Compartment containing sugar modifying enzymes
• trans-


  Compartment containing sugar modifying enzymes
• Glycan modification process


  1. Sequential, the product of one later is the substrate for the next

  2. Akin to an assembly line
• Cis-Golgi glycans


  Substrates for medial golgi enzymes
• Medial-golgi glycans


  Substrates for trans-gogi enzymes, then exit
• "Cisternal maturation"


  The glycoproteins do not move from one compartment to another, rather, the compartment itself matures, i.e, the cis face cisternae mature and move towards medial and then trans face, and replaced by newly formed cis-cisterna
• Congenital defects of glycosylation


  • Heterogenous disease resulting from deficiencies in glycan modifying enzymes in golgi compartments
  • Reduced branching and glycan extension
  • Characterized by wrinkly skin, muscular dystrophy, etc
• Mitochondria


  • Multi membrane organelle
  • Site of aerobic oxidation: convert nutrient + O2 to ATP + CO2 + H2O
  • The cell's combustion engine
  • Plant equivalent: chloroplast
• Mitochondrial DNA


  • Size and coding capacity vary between organisms
  • Always code mitochondrial proteins, the rest coded by nuclear DNA
  • Inherited cytoplasmically
  • Inherited maternally
• Mitochondria


  • Organelle or endosymbiont (evolved by from bacteria that were endocytosed by ancestral cells)
  • Most bacterial genes were lost overtime, leaving only genes that gave cells an advantage
  • Can undergo fission like bacteria
• Mitochondrial membranes


  • Outer membrane - smooth
  • Inner membrane

  
• Mitochondrial morphology

  • Can vary from individual spheroid/ovoids to long elongated networks
  • Related to balance of fusion and fission events