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Cell Structure - Coggle Diagram

- - - - BiP can bind transiently to nascent polypeptide chains as they enter the ER during contranslational translocation
      - BiP prevents segments of nascent chain from misfolding
      - calnexin and calreticulin selectively bind to some N-linked oligosaccharides on growing chains (its ligand formed by glucosyltransferase), binding of these 2 to unfolded chains marked with glucosylated N-linked oligosaccharides prevents aggregation
      - Peptidyl-prolyl isomerases accelerate rotation about peptidyl-prolyl bonds at proline residues in unfolded segments
      - unfolded-protein response
        
        Ire1 is an ER membrane protein that exists both as a monomer and dimer
        
        this is the stress response that responds to accumulation of unfolded protein in ER by activating enzymes involved in protein folding
        
        the dimeric form promotes Hac1 formation that activates expression of genes induced in this response, but the dimer formation can be prevented by BiP
        
        Another response to unfolded proteins is proteolysis of ATF6 (transmembrane in ER), its cytosolic domain is released by proteolysis and moves to nucleus to promote chaperone transcription through Notch signalling pathway
        
        Studeis have shown that misfolded secretory proteins are recognised byspecific ER membrane proteins and are targeted for transport from ER lumen into the cytosol by dislocation
        
        dislocation
        
        recognition involves trimming of Nlinked carbohydrate chain by mannosidases in Er which are recognised by OS-9, those with no oligosaccharide can be targeted suggesting other processes
        
        ERAD complex enables dislocation of misfolded protein, likely by Sec61 translocation channel repurposed
        
        as segments of dislocated polypeptide emerge into cytosol p97 uses ATP to pull into cytosol where ubiquitin ligase of ERAD add ubiquitin
    - - They are formed by oxidative linkage of sulfhydryl groups on 2 cystein residues catalysed by protein disulphide isomerase (PDI) (abundance in ER of secretory cells)
      - Disulphide bonds in PDI active site can be reasily transferred to protein by 2 sequential thiol disulphide transfer reactions which generates a reduced PDI (regenerated by Ero1 which carries a disulphide bond that can be transferred to PDI)
      - Proinsulin has 3 disulphide binds that link Cys1 and 4, 2 and 6, 3 and 5
    - - proteins are glycosylated (Asparagin(N)-linked glycosylation makes proteins hydrophilic to stop aggregation and helps folding) (see 13.3)
      - Begins with oligosaccharide precursor with 14 residues, which is then added to protein
      - They are then modified by changing up to 9 of the 14 residues
      - Prior to transfer to polypeptide chain the precursor is assembled on dolichol phosphate (attached by pyrophosphate bond), the 3 glucose residues are the last added during synthesis and act as signal
      - The 14 residue precurosr is transferred from dolichol to asparagine residue (in Asn-X-Ser and Asn-X-Thr only since needs oligosaccharyl transferase) on nascent polypeptide
      - Immediately after this transfer glycosidases remove the 3 glucose residues and on mannose residue
  - - - Type I
        
        single pass
        
        cleaved at N-terminus
        
        C-terminus on cytosolic face, N
        
        all type I proteins contain approximately 22 amino acids that become membrane spanning a helices
        
        Once N terminus enters, SP is cleaved and growing chain continues but when transmembrane domain passes translocon its passage stops and the domain laterally moves into membrane
        
        The hydrophobiciity of this segment anchors it into membrane and is called the stop-transfer anchor sequence
        
        synthesis of polypeptide continues but not through translocon but into cytosol
        
        HGH receptor is typical type I insertion and mutants with charged residue have translocation entirely into ER
      - Type II
        
        internal sequence is after a positive amino acid side chain
        
        not cleaved signal sequence
        
        N terminus on cytosolic
        
        possess a single internal hydrophobic signal anchor sequence, in type II this directs insertion of nascent polypeptride into ER membrane so N terminus faces cytosol using type I mechanism
        
        However internal signal anchor sequence is not recognised by signal peptidases so not cleaved, the signal anchor sequence can move laterally into bilayer where it functions as anchor
        
        as elongation continues the C terminal region is extruded through translocon into ER lumen
      - Type III
        
        use internal signal sequence so no cleavage
        
        positive amino acid side chain is after the signal sequence
        
        C-terminus on cytosolic
        
        This also have a single internal hydrobic signal anchor near the N terminus and directs insertion of nascent chain into ER mebrnae with N terminus facing lumen
        
        the signal anchor sequence also functions like a stop transfer sequence and prevents further extrusion of elongating chain into ER lumen
      - Type IV
        
        internal signal sequence binds to SRP which binds to receptor which targets translocon
        
        signal sequence is not cleaved
        
        positive residues position determines orientation in translocon
        
        N terminus in cytosol
        
        examples- glucose transporters and most ion channels
        
        hydrophobic segment close to N terminus is inserted into ER membrane
        
        as nascent chain following transmembrane segment elongates it moves through translocon until second hydrophobic segment is formed
        
        This helix prevents further extension of nascent chain through translocon so function similar to stop transfer anchor sequence
        
        C terminus in cytosol
        
        example- G protein coupled receptors (contain 7 a helices)
        
        the hydrophobic transmembrane segment closest to N temrinus is often folowed by cluster of positively charged amino acids so nascent polypeptide is inserted into translocon with N terminus extending into lumen
- - - - at endosomal pH (5-5.5) His residues in B-propeller domain of receptor become protanted causign high affinity to the Cys repeats in LDL binding domain
      - this causes release of bound LDL particles and the LDL receptor is efficiently recycled back to the plasma membrane
  - - - synamin forms a spiral structure around neck of budding vesicle
      - GTP hydrolysed which narrows the spiral pulling the membranes closer which excludes water allowing hydrophobic regions to fuse
      - mutants of dynamin form buds but don' pinch off
  - - - VAMP (the v-SNARE) is incorporated into membraen as bud from trans-Golgi network, and syntaxin and SNAP-25 (t-SNAREs) are attached to membrane by lipid anchor
      - The 4 helix bundle forms as 1 from VAMP, 1 from syntaxin adn 2 from SNAP-25 coil around each other
      - the embedded hydrophobic transmembrane domains of VAMP and syntaxin pull the vesicle to target membrane
      - the energetically favourable formation of 4 helix bundle can overcome electrostatic repulsion of negative phospholipid heads allowing fusion
- - - - cargo receptor recruits cargo
      - Sar1 recruits adaptors to receptor
      - adaptors (Sec23/24) bind receptors, membrane cargo proteins are recruited to vesicle using the Sec23/Sec24 complex
      - Coat proteins(Sec13/31) bend membrane
      - Sar1 binds to GTP and triggers COPII vesicle coat assembly (Sar1 is also only GTPase switch protein activated in ER membrane so only COPUU bud from ER)
      - Sec12acts as a GEF for Sar1 so GDP is released and binds GTP, this GTP binding causes change in Sar1 which exposes amphipathic N terminus which embeds itself in bilayer
      - After vesicle coat formed, Sec23 coat subunit promotes GTP hydrolysis by Sar1 and release of SarGDP from vesicle membrane disassembles coat
    - - ARF protein is a GTP binding protein and undergoes similar nucleotide exchange and hydrolysis
      - myristate anchor (covalent modification) on N terminus of ARF tethers it ARFGDP to Golgi which changes ARF conformation
      - The change means hydrophobic residues in N terminal segments insert into membrane bilayer and the tight association allows coat assembly
  - - - COPII formation triggered when Sec12 exchnages bound GDP for GTP in Sar1, this exchange induces Sar1 binding to ER binding and to Sec23/Sec24, this then allows binding of Sec13/Sec31 to form COPII
      - Sec13/Sec31 spontaneously assemble into cagelike structures
      - Sec16 (ER cytosolic protein) interacts with Sar1GTP to organise coat proteins and increase polymerisation efficiency
      - ER membranes can be recruited to COPII, many containing di-acidic sorting signals whihc bind to Sec24 on COPII coat and are involved in selective export of certain proteins from ER
    - - COPI coat is formed from coaromers (cytosolic complexes made of 7 polypeptide subunits
      - Most ER resident proteins carry Lys-Asp-Glu-Leu sequence at C terminus (this is necessary for protein location by the KDEL receptor (a transmembrane protein found in vesicles between ER and cis-Golgi and cis Golgi membrane))
      - The KDEL receptor and other membrane proteins are transported back to ER using Lys-Lys-X-X sequence that binds to COPa and COPB
    - - cisternae compartments fidder from one another based on the enzymes they contain, and it is now known that the small vesicles in the vicinity of the Golgi are mivigng in the retrograde direction so Golgi has a highly dynamic structure
      - evidence
        
        microscopic analysis of algae scales synthesis revealed that they are assembled from glycoproteins in cis into large dense complexes that then move to trans, but were never obsevered in vesicles
        
        IN fibroblasts large procollagen precursors often form in cis lumen bjt are too large to be incorporated into vesicles
        
        fluorescent labels of cis and trans Golgi resident proteins revealed that few compartments ever contained both proteins but over time cis Golgi progressively lose this protein and acquire trans
    - - The best characterised vesicle contains outer clathrin layer and adaptor protein complex inner layer
      - the APs determine which cargo are included in budding transport vesicles by binding to cytosolic face of membrane proteins and all coats containing an AP complex uses ARF to initate assembly
      - APs
        
        when going to lysosome, the clathrin coat is assembled with AP1 or GGA. AP1 binds to proteins with Tyr-X-X-Φsequence, and GGA binds to Asp-X-Leu-Leu and Asp-Phe-Gly-X-Φ
        
        some contain AP3 complex which has no clathrin binding site, they still transport to lysosome but bypass late endosome and fuse directly
      - Pinching off
        
        dynamin polymerises arounf the neck of the bud and then hydrolyses GTP (which provides energy to drive conformational change)
        
        cytosolic Hsp70 (chaperone) uses energy from ATP to drive depolymerisation of clathrin coat into triskeletons and exposes v-SNAREs for use in fusion with target membranes, cytosolic Hsp70 uses auxillin (co-chaperone)that stimulates ATP hydrolysis
      - To lysosomes
        
        mannose 6-phosphate (M6P) is formed in cis-Golgi and is sorting signal to direct lysosomal enzymes from trans to late endosome
        
        In cis N-linked oligosaccharide on most lysosomal enzymes undergoes process to generate M6P residues on oligosaccharide chains whihc prevents further processing
        
        transmembrane M6P receptors bind the M6P residues on lysosome destined protein, Clathrin/AP1 vesicles (containing this receptor) then bud from trans Golgi, lose their coat and fuse with late endosome
        
        A phosphatase within late endosome usually removes phosphate from M6P on lysosomal enzymes preventing rebinding to M6P receptor adn retromer recycle the receptor back into trans
    - - Proteolytic processing
        
        some only have N-terminal ER signal peptides removed from nascent chain but some membrane proteins and soluble secretory proteins are synthesised as precursors (called proproteins)
        
        proproteins require further processing to generate mature, active proteins and this conversion usually occurs after being sorted into vesicles
        
        proenzymes are sorted by M6P receptor and have delayed activation since they would otherwise digest other components in secretory pathway
        
        proproteins of most constitutively secreted proteins are cleaved only once at a site C-terminal to a dibasic recognition sequence like Arg-Arg or Lys-Arg by endoprotease (examples- furin processes albumin, PC2 and PC3 processes hormones)
      - sorting into membranes
        
        In one mechanism the sorting takes place in trans network, the different vesicel types for apical and basolateral can be distinguished by protein constituents (like Rab adn v-SNARE)
        
        mutational studies on VSV G protein that are specifically targeted to basolateral have defined tyr-X-X-Φ and Asp-X-Leu-Leu sorting signals which are recruited into clathrin/AP coated vesicles so clathrin coated vesicles are involved in basolateral membrane
        
        GPI cells are usually targeted to apical membrane, however GPI anchor is not an apical sorting signal in polarised cells and can go to basolateral in thyroid
        
        Another mechanism for sorting apical and basolateral proteins acts in hepatocytes. Both types are transported in vesicels from trans to basolateral by exocytosis
        
        From there both basolateral and apical are endocytosed in the same vesicles but then paths diverge (basolateral sorted into vesicle adn recycled back into basolateral, and apical are sorted into vesicles and move across cell by transcytosi)
- - - - if the object is closer than the focal point then the image will be enlarged
      - decreasing the focal length produces a larger virtual image (aka increases magnification)
      - if the object is further than the focal length a real image is formed
    - - an objective lens produces a magnified real image
      - an eyepiece lens produces a magnified virtual image of the real image
  - - - different parts can be stained using specific dyes or antibodies
      - e.g. DAPI slides in between base pairs and is blue
      - light of a specific wavelength excites fluorophore which then emits light of a longer wavelength
      - its resolution can be improved using confocal microscopy or deconvolution which removes the out of focus info
      - the microscope allows excitation light to pass through objective lens into sample and allows observation of light back through objective lens by using a dichroic mirror
      - Ion sensitive
        
        Fura-2 is a fluorochrome that binds to single Ca2+, its binding increases the fluorescence of Fura-2 at one wavelength, at a second wavelength of Fura-2 there is no differences if bound or not, measuring changes in ratio of fura-2 fluorescence at 2 wavelengths can reveal Ca2_ concentration
        
        fluorescent dyes sensitive to H+ reveal pH, other pribes use a fluorochrome linked to weak base that is only partially protaonated at neutral pH so can freely permeate membranes and in acidic conditons the probes become protonated and protonated probes cannot recross membrane so accumulate
      - FRET is a technique used in which emission wavelength of first is close to excitation of second wavelength. The efficiency of FRET is proportional to (distance between fluorochromes)^-6. FRET biosensor contains 2 fluorophores on a single polypeptide, and when meets signal undergoes change bring close together and generating FRET
    - - primary antigen binds to antigen on cell structure
      - secondary antibody with colour (, fluorescent dye covalently attached to antibody) binds to primary antibody
      - cell must be fixed and made permeable to antibody entry by non-ionic detergent or extracting lipids with organic solvent
      - double-label fluorescence microscopy- 2 proteins visualised simultaneously, for example 2 proteins visulaised by indirect immunofluorescnce using primary antibodies made in different animals and secondary antibodies labelled with differnet fluorochromes
      - cDNA encoding a recombinant protein fused to epitope tag enters cell and encodes recombinant protein linked to specific tag (common tags are FLAG and Myc) these can be used to detect the recombinant proteins in the cell
      - using recombinant DNA technology, GFP coding sequence is fused with coding sequence of protein and so protein is tagged, protein is often unchanged by GFP so protein dynamics observed
      - major limitations are fluorescent light emitted comes from plane and molecules above an dbelow it creating blurred image and visualisation of thick specimens requires images at multiple depths and then alligned
      - deconvolution microscopy
        
        uses computational methods to remove fluorescnce of out of focus sample planes, to do so make series of images of focal plane from a test slide containing tiny fluorescent beads
        
        the beads represent point of light object becomes blurred outside focal plane so used to determine point spread function to enable calculation of points of blurriness
        
        once calibrated the experimental series of images can be deconvoluted
      - Confocal microscopy
        
        This uses optical methods from specific focal plane and excludes light from other planes
        
        there are 2 types point-scanning and spinning disc confocal microscope, both use emitted fluorescent light from one small areia of focal plane, so out of focus light is excluded, by collecting light through pinhole before reaches detector, illuminated area is then moved across whole focal plane to build image
        
        difference- pointscanning uses a laser light source at excitation wavelength to scan focal plane in raster pattern, collect emitted fluorescence in photomultiplier tube, however this is a long process so not good for very dynamic cells, and it illuminates each spot with intense light which can bleach fluorochrome. Spinning disc uses excitaion light from laser and spreads it to illuminate small part of disc spinning at high speed with pinholes that are used to scan focal plane of sample, emitted light returns through pinhole and reflected by dichroic mirror and focused onto camera; limited by pinhole size which is fixed and has to be matched to objective lens magnification
    - - used to study molecular mobility
      - cell is labelled with GFP (fluorescent reagent)
      - a lazer bleaches a certain area of membrane (photobleaching) and the image changes over time
      - diffusion coefficient can be measured
    - - 3D structure illumination doubles resolution
      - extra resolution is calculated from inference patterns
      - types
        
        structured illumination microscopy- specimen illuminated with pattern of light adn dark stripes and images are taken as rotated; analysis gives 100nm resolution
        
        Stimulated emission depletion- sample scanned like point-scanning but laser point surrounded by donut shaped depletion beam making area excited much smaller; computer records position of the spot excited and records emission and builds up image
        
        photoactivated localisation microscopy- uses GFP variant photoactivation so it becomes fluorescent only after excited by specific wavelength (different from excitation wavelength), a small percent of GFPs are activated and each localised with high precision and this repeats until a high res image forms
    - - used to locate single fluorophore molecules which is then reconstructed to form super resolution images
      - comparison of images over time can be used to calculate an average
    - - TEM
        
        uses 2D projection image of a thin specimen
        
        requires a vacuum since atoms in air absorb electrons and specimen needs to be thin
        
        electrons are emitted from a filament and accelerated in an electric field, condenser lens focuses elctron beam onto sample, objective and projector lenses focus electrons that pass through specimen and project them onto viewing screen
        
        Preparation
        
        To view single macromolecules, sample first prepared by absorbence to a 3-mm electron microscope grid, coated in a thin carbon, plastic film, sample then bathed in heavy metal solution and excess solution removed; the solution coats the grid but excludes regions where sample is adhered, and when viewed the areas where no stain is seen so negatively stained
        
        Other way is to absorb sample to small piece of mica, then coat with thin platinum film and dissolve in acid or bleach' when transferred to grid adn examined it shows 3D topology (called metal shadowing)
        
        Thin sections- chemically fix samples, dehydrate, impregnate with lipid plastic to harden then cut 5-100nm thick
        
        Cryoelectron
        
        an aqueous suspension of sample applied to grid in extremely thin film, frozen in liquid nitrogen and maintained in state using a mount
        
        frozen sample is then plasced in electron microscope at very low temperature to prevent water evaporation and so the sample can be observed in detail without heavy metal staining
        
        computer based averaging of many images can develop a 3D model
        
        cryoelectron tomography is an extension allowing determination of organelles adn cells in ice using images from different angles, a disadvantage of this is samples must be 200nm then (compared to 200um for confocal)
      - SEM
        
        the electrons collected either emitted (secondary electrons- high energy from sample) or reflected (back scattered) that are focused on cathode ray
        
        surfaces are observed of metal coated specimens
        
        the resulting micrograph has 3D appearance because secondary electrons produced by any point on surface depends on angle of electron beam in relation to surface
        
        resolving power limited by thickness of metal coating (10nm)
    - - generates image in which degree of darkness/brightness depends on refractive index
      - a cone of light generated by an annular diaphragm in condenser lens illuminates specimen, lihgt passes through specimen into objective lens and unobstructed light passes through phase plate that transmits small percent of light and chages its phase
      - the light that passes through specimen is refracted and will be out of phase with part that did not pass through
      - how much phases differ depends on differences in refractive index , all the ligh tis then combined in image plane to form image
    - - Focus a cone of lasar light on a spot that scan across focal plane (this does generate out of plane focus but removed by collecting light through pinhole), but does cause photobleaching and phototoxicity damage
      - to remove these problems, a fluorophore can be excited by 2 photons of half energy (double wavelength) so the laser cone is focused on spot in one plane so only at focal point is there enough density to excite fluorophore
      - the lower energy also means no out of focus signal and less photobleaching or phototoxicity
    - - During this only the portion of specimen immediately next to coverslip is illuminated so there is minimal out of focus background
      - The excitation light comes through objective lens but the angle it arrives is changed to be slightly larger than critical angle so light is reflected off coverslip
      - This generates narrow band of light called evanesent wave that illuminates only about 50nm of sample next to coverslip
      - used in identifying structures on bottoms of cells grown on coverslip and measuring kinetics of assembly if microtubules and actin
  - - - histological samples are often stained with hematoxylin and eosin, hematoxylin binds to basic amino acids and eosin binds to acidic molecules
      - teh dyes stain various cell types sufficiently differently so they can be distinguished
- - - - they are called karyopherins or importins/exportins
      - Importin α/β carry classical NLS containing proteins
      - CRM1 is a major protein export factor, TAP is an export for mRNA
      - They also have affinity for FG-repeats, because of this the receptors and bound NLS-containing cargo proteins partition into fluid phase in central NPC channel
      - nuclear transport receptors bind NLS on a cargo protein to be transported into nucleus
      - Once inside the channel the importin-cargo complex can reach the nuceloplasmic side of channel and the importin interacts with Ran-GTP
      - this interaction causes conformational change in importin to displace NLS and release the cargo protein into nucleoplasm
      - Importin-RanGTP complex diffuses back through NPC, then Ran interacts with GTPase activating protein to stimulate RanGTP hydrolysis to GDP, causing low affinity for importin so importin released into cytoplasm
      - RanGTP travels back through pore where it encounters Ran-GEF causing Ran to release GDP for GTP
    - - Ran is a molecular switch that exists in either a GTP or GDP bound conformation
      - RanGAP stimulates Rab, because it has low intrinsic GTPase activity, and hydrolyses GTP
      - RanGEF causes the release of GDP from Ran allowing GDP to rebind
  - - - structural
        
        form scaffold of nuclear pore which is a ring of eightfold rotational symmetry that transverses both membranes of nuclear envelope to create opening
        
        connected to membranes by highly curved region of membrane that contains embedded membrane nucleoporins (the other type)
        
        7 nucleoporins form Y shaped structure called Y complex, 16 copies of the complex form basic structural scaffold
      - FG-nucleoporin
        
        line the channel of NPC and also found associated with nuclear basket
        
        they contain multiple repeats of short hydrophobic sequences which occur in regions hydrophilic chains
        
        they are essential for NPC funciton but depends on general property of these proteins rather than structure since NPC remains functional if FG repeats are deleted
  - - - 3 types
        
        leucine-rich sequence found in PKI adn in the Rev protein of HIV
        
        Other 2 recognised in different heterogenous ribonucleoprotein particles
      - They are a nuclear-export signal that stimulates their export from the nucleus to teh cytoplasm
      - a specific nuclear transport receptor (exportin 1) forms complex with RanGTP in nucleus and then binds the NES in a cargo protein
      - binding of exportin 1 to RanGTP causes change in exportin 1 that increases affinity for NES so a trimolecular cargo complex forms
      - exportin 1 then transiently interacts with FG repeats in FG nucleoporin and diffuses through NPC
      - cargo complex dissociates when it encounters the RanGAP associated with NPC cytoplasmic filaments stimulating hydrolysis of bound GTP which lowers exportin 1 affinity
      - RanGDP dissociates from trimolecular cargo complex, exportin lowers affinity for NES whihc releases cargo into cytosol
      - exportin 1 and RanGDP are then transported back into the nucleus through the NPC
    - - once completely processed, mRNA remains associated with proteins in a messenger ribonuclear protein complex (mRNP)
      - principal transporter of mRNPs is the mRNP exporter (composed of nuclear export factor 1 and nuclear export transporter1), multiple of NXF1/NXT1 dimers bind to mRNPs through cooperative binding
      - both NXF1 and NFT1 interact with FG repeats which allows diffusion through the central channel of the NPC
      - Once mRNP-NXF1/NXT1 complex reaches cytosol the dimers dissociate from mRNP using RNA helicase Dbp5