Plant Cell Biology PDF

Fiche résumé TD biocell Microscopy Light microscopy Visible light : 400(violet) to 800(red) (w/ cyan, green, yellow, orange in btwn) Cell : fundamtal unity of life (self reproducing) : 1 cell ~ 10µm (biggest cell : oocyte: 0,1 mm). Wide ce...

Fiche résumé TD biocell Microscopy Light microscopy Visible light : 400(violet) to 800(red) (w/ cyan, green, yellow, orange in btwn) Cell : fundamtal unity of life (self reproducing) : 1 cell ~ 10µm (biggest cell : oocyte: 0,1 mm). Wide cell variety : in size, function, behaviour and pluricell or unicell beings Differenciation of cells : Totipotent cell (capable of producing a full organism), pluripotent cell (=stem cells). Specialization (wide variety of biological functions). Subcellular molecules & structures (plasmic mb, cytoplasmic compartmt, DNA) Procaryotes (unicell beings)(1 to 2µm) / Eukaryotes (true nucleus & nuclear envlp)(5 to 100µm) / Virus 50 to 100µm Plasmic mb thickness : 7nm or 70A° and 70 atoms of hydrogen; proteins (100A°) Fiche résumé TD biocell 1 Fiche résumé TD biocell 2  Separating power of the microscope formula : n is the refractive index btwn the objective and the object and a is the digital aperture of the lens the bigger epsilon is ; the lower the resolution ⇒ You can see better at smaller wavelengths ~ 400nm (violet) Diff types of light microscopy Light-field microscopy : Use thin slices of tissue or cells ⇒ direct transmission of light (visible light or uv light) through the sample ⇒ observation of colored objects (stains) (expl : HES stains basic environnemt - stains dna and histones) Black field microscopy : light rays pass laterally over the object ⇒ a strong refractive index is needed (areas that diffuse light appear shiny) Phase contrast microscopy : Dense or thick part of the cell = delayed light (~gives the impression of a 3D image due to the contrast) ⇒ observation of living cells Differential interference contrast microscopy : uses polarized light (vibrates in only 1 direction) : gives a 3D vision of a translucent object Sample preparation : Living cells : cell culture = ex-vivo (have to maintain their ability to divide) Culture primary in vitro / immortalized cell lines / Organ slices (cells are alive for a fex hours). Growing conditions : sterile, in suspension (nutrients + serum)& in incubators (37°C) Fixed cells : dead cell ketp as close as its living state. Physical fixation : heat fixation or freezing. Chemical fixation (use of fixators). Light has to go through sections of the sample (cut with diamond blade (=microtome) ⇒ solidify by dehydrating & replace water by paraffin& ethanol as a mediator. Then dewaxing and progressive rehydratation, than staining and new dehydratation Fiche résumé TD biocell 3  Electron microscopy Smallest human cell: erythrocyte (red blood cells): 5µm &Cell mb :plasmalema : 7 nm Electrons are smaller than photons ⇒ resolution of the order of a nm ⇒ max magnification of light microscopy (X1000) / elctron microscopy (X1K to X500K). Charge of an electron : 1,6x10-19 C Electrons are derived from a filament of tungsten brought to high temperature emitted under the vacuum. Advantages : ⇒ separeting power < nm. ⇒ We can see inside of the cell (organelles..) Constraints : Samples dehydrated bc of vacuum + are ultra thin + no living cells (bc of water) Sample preparation Fixation Fiche résumé TD biocell 4  Physical fixation by freezing: avoid formation of ice crystals who would break the cells (liquid propane: -196°C or Helium: -272°C) L> impossible to maintain while observing on the electronic microscope bc the electrons will heat up the sample Chemical fixation: Maintains the macromolecular structure existing already in the cells: Stabilizes and consolidates the cell structure - OsO / Tetroxide osmium: Creates links with ethylene bonds -Glutaraldehyde: Form bridges with groups amines from protein (Possibility of double fixation using glutaraldehyde) Conditions : Sample have to be 50 to 80nm + preparation must be solid (in resin + cut with idamond knife) + sample on a copper grid and covered with heavy metals Fiche résumé TD biocell 5  Cytochemistry ⇒ study of chemical constituants of the cell (proteins, lipids, nucleic acids, glucids) + identification & localization of chemical contiutants in cell Protocol of creating a sample : Stain ⇒ fixation (chemical /physical) ⇒ dehydratation ⇒ put in wax / resin ⇒ cut (((not sure abt that))) Staining : specificity : chemical constituants must b intact 1 specific to that compound & sensitivity : chemical constituant in the smallest qtity possible. Avoid overstaining or lack of staining Fixation (cytochemical reaction are done ON the slices) : Insoluble compounds: Stay following fixation > Are detectable Hardly soluble compounds: Detection depends on fixation Soluble compounds: e.g. glucose: undetectable (leaves ith the water during dehydratation) (but we can detect glycogen) Fixator shouldn’t solubilize the compound (like ethanol). Waxing above 60°C : bad for enzyme ⇒ freezing is better to fixate the cell Sugars : Fiche résumé TD biocell 6  Source of nrj : most common : glycogen alpha glycol bond : btwn 2 carbon atoms alpha 1,4 bond : bond btwn two adjacent sugars (glucoses) Association of sugars with proteins = glycoproteins which are a major part of the membrane proteins (expl : glycocalyx at the surface of cells) Association of sugars with lipids = glycolipids which are a major part of the membrane lipids Sugars are the main part of nucleotides. Glucose cannot be detected naturally because of its solubility, however we can detect its insoluble form > Glycogen Reactions PAS reaction : to detect sugars (periodic acid and Schiff reagent) ⇒ Periodic acid will hydrolyze the a-glycol bonds of the glycogen via releasing the aldehydes groups Once the aldehyde groups are released, the Schiff’s reagent will be applied Schiff’s reagent contains Fuschine which emits red color while being fixed to the aldehyde groups Lack of specificity of the reaction : to be sure we have glycogen : alpha- amylase control : breaks the 1,4 bonds of glycogen ⇒ no detectable unsoluble form Fiche résumé TD biocell 7  - Slice treated with PAS: >> Staining - Slice treated with PAS + alpha-amylase: L> If staining is produced: molecules other than Glycogen were stained L>No staining : It was Glycogen Feulgen reaction : stain DNA (nucleotides : Phosphoric acid coupled to nucleoside) L> Only DNA is stained → Specific oxidation by HCL = hydrochloric acid → DNA is hydrolyzed and releases puric bases → Aldehyde groups of sugar are released → Schiff’s reagent contains Fuschine which emits red color while being fixed to these aldehyde groups Toluedin Blue : stain nucleic acid (basic dye) Problems : No difference btwn RNA and DNA + it can also fix proteins when AA are negative ⇒ work with toleudin at acid pH so only nucleic acids will b stained. Control of reaction : Hydrolization of nucleic acids with HCl→ destroyed If staining is produced: no Nucleic Acids stained NO staining : Nucleic Acids were stained Fluorescence microscopy Definition: Fluorescent molecule : fluorophore or flurochrome. When an atom/mol absorbs light and re-emits it simultaneously = it is fluorescence Fiche résumé TD biocell 8  Fluorescence: most used / Phosphorescence (might be used in electron microscopy for the screen or in phosphorescent sticks) / Bioluminescence (made by an enzyme) Law of energy : UV has the highest energy ⇒ the lower the wavelenght, the more energy it has Excitation filter has to let pass only the color that we want to excite (if we want red, we need a filter that does not let pass any color with a bigger wavelength than green) In cells → components that are capable of autofluorescence : amino acids, proteins.. Fluorophores Fiche résumé TD biocell 9  Fluorophore selection : 3 sorts of fluorophores DAPI ; stains dna in a very dark blue FITC : emits green color (stains les fuseaux mitotiques) Rhodamine : emits red color To obtain a certain color, you have to excite another color with a smaller wavelength (to get a green emited color, you have to excite blue) DAPI : Intercalating agent FITC : in mushrooms, we can find phalloidine that can bind to microtubules ⇒ we can combine it to FITC to obtain fluorescent microtubules Methods : PCR, Enzymes (specifitcity), Antibodies : extra extra specific Fiche résumé TD biocell 10  Image 1 was obtained with DAPI ⇒ obtained by an intercalating agent (DAPI) Image 2 ⇒ obtained with FITC ⇒ obtained by natural proteins like phalloidine (or antibodies) Image 3 ⇒ obtained with rhodamine ⇒ obtained by antibodies coupled with fluorophore Antibodies as fluorophore carriers We are able to detect fluorescence thanks to the fixation of secondary antibodies bc w/ only 1 fluorophore and 1 primary antibody you can’t see well Fiche résumé TD biocell 11 Fiche résumé TD biocell 12  Primary antibodies have to be another species than the antigen bc the immune systm doesn’t normally attacks its own antigens ⇒ same for the secondary antigen that cannot be from Fiche résumé TD biocell 13  the same species as the 1st antigen. Sme principle as ELISA technique Plant cell biology Plant cell organisation 1. Cytoplasm 2. Cell wall (outer membrane : plasmalemna) 3. Vacuole (w/ tonoplast : inner mb) Fiche résumé TD biocell 14  4. 5. Mitochondrion (w/ an evlp surrounding it) 6. Cytoskeleton (w/ microtubulates (tubulin) and microfibers ( actin) 7. Nucleolus (RNA → ribosomes) 8. Nucleus 9. Nuclear enveloppe 10. Reticulum 11. Peroxysome (digestive enzymes usually for protein ) 12. The golgi apparatus (maturation of proteins) 13. Plasomdesmata (plasmodesme : petit trou de communication~ pore) 14. Chloroplast (w/ 2 mb) 15. Ribosoms 16. Globules of lipids Plant cell wall Cellulose microfibrils : cellulose : polymer of glucose, beta(1-4) glucan ⇒ 20 polymers → 1 cellulose microfibril : stacked // on the same layer at diff angles Middle lamella : contains pectin : homopolymer of galacturonic can associate w/ other mol. 1ary cell wall : Very thin cell wall / 2ndary cell wall : Thick & lignified & cellulosic. Identification of cell wall compounds Glacturonic acids stained : light pink. Periodic acid reaction : induces aldehydes on glucose residues ⇒ schiffs reagent → cellulose becomes fushia = everywhere except the middle lamella. Vacuole : Cell is at equilibrium w/ environnement (slightly hyoptonic) ⇒ turgid state If cell in hypertonic : water goes out + solutes go in ⇒ equilibrium Fiche résumé TD biocell 15  Cytoplasmic tractus ⇒ when ruptured ⇒ cell is going to die for sure Plastids Chloroplasts 2 mb ⇒ the enveloppe : 1 mb → very similar to plants and other is very similar to the mb of procaryotes. (11 & 12) Bigger thylakoïds and smaller thylakoids ⇒ granum (9) 8 → diffuse genetic material : extranuclear genetic material Intraplastidial starch (1) Fiche résumé TD biocell 16  Plastids move around the vacuole. Stomata (guardian cells) opens with ATP & accessory cells might have a nucleus and leucoplasts Chromoplasts ⇒ have 0 thylakoids and a disorganised inner mb (=stromule) Amyloplast (diff from starch granules ⇒ the amyloplast has a mb and contains the starch granules). They differ according to plant families (inside → sort of crystal of nucleus : the hilum with multiple circles (like shockwaves) = lines of stratification Fiche résumé TD biocell 17  Starch grain has 2 polymers : amylose (linear polymer ) and amylopectine (structure like a coral bc of aplha 1-6 liaisons btwn 2 amyloses) ⇒ starch has alpha liaisons 1-4 (=/ cellulose who has beta liaisons) Mitosis Mitosis → to grow / meiosis : to produce gametes. Mitosis : conformal multiplication of the mother cell → diploid lineage. Chr duplicate and chromatides migrate. Meiosis : chromatic reduction → haploid phase. Fiche résumé TD biocell 18  Chromatin : DNA condensed in a specific oragnisation xith histones Heterochromatin : Very condensed : replicated late Euchromatin : Less condensed : replicated first; genes regularly transcribed Nucleolus : Histones octomers + Condensed DNA Plant mitosis Roles of roots : Absorption of water + mineral salts, via root hairs of the hair zone + Anchoring in the ground Root auxesis zone = with Auxin elongation of the cell (⇒ rectangular shape) + differenciation → cells acquire diff phisiological functions Root meresis zone = daughter cells in a specific position = mitotically active pluripotent stem cells = meristem Stages of mitosis 1st→D → nucleolus & barely condensed DNA (pre prophase) 2→ B : prophase (no nuclear envlp or nucleus) Fiche résumé TD biocell 19  3→ G : centromers are aligned on the equatorial plane (metaphase) 4→ A : chr are starting to move away from each other. 5→ E : chr reach the polar cap 6→ C : start of decondensation - fragmoplast 7 → F : single mass of DNA Cytokinesis is prepared throughout mitosis. The division plane is determined since the prophase thanks to the pre- prophase band (PPB,*). The site is identified using specific proteins and microtubules. Golgi vesicles (wall material) are transported via the cytoskeleton towards this plane, and fuse in a centrifugal manner (arrow). They form the cell plate. This specific zone of the cytoskeleton is called phragmoplast Animal mitosis No cell wall to divide cells (centrioles at the caps of cell : star shaped filaments) Contractile ring : constricts the mother cell in the middle Fiche résumé TD biocell 20 Fiche résumé TD biocell 21