國(guó)外大學(xué)細(xì)胞生物學(xué)課件英文5bioy

1、國(guó)外大學(xué)細(xì)胞生物學(xué)課件英文5bioy國(guó)外大學(xué)細(xì)胞生物學(xué)課件英文5bioyW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 2W2011 D103 - lecture 5 SuettW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 3Does a cell have a skeleton?W2011 D103 - lecture 5 SuettWhy do cells need a skeleton?Cells n

2、eed a (cyto)skeleton to:Generates and maintains shapePromotes movement of a cell within a cell4W2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. Why do cells need a skeleton?CCytoskeletal elementsW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 51. Actin (m

3、icrofilaments)Cytoskeletal elementsW2011 D10Cytoskeletal elementsW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 62. Intermediate filamentsCytoskeletal elementsW2011 D10Cytoskeletal elementsW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 73. Microtubules

4、Cytoskeletal elementsW2011 D10How can the cytoskeleton change in response to external signals? W2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 83.Site and rate of filament nucleation is critical and tightly regulated.Assembled from small subunits,subunits are not covalently li

5、nkedRegulated polymerization/ depolymerizationHow can the cytoskeleton changAssembly of cytoskeletal elements into higher order structures W2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 9Assembly of cytoskeletal elemeThe actin cytoskeleton10The actin cytoskeleton12actinG-acti

6、n assembles into F-actinG-actin (monomeric building block) assembles into F-actinpolarized helical filament of 6 nm in diameterThe addition of ATP-bound G-actin to a filament requires ATP (energy requirement)W2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. actinG-actin assemble

7、s into F-Filament nucleation is the rate-limiting stepW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 12Filament nucleation is the ratActin filaments undergo treadmillingW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 13faster growing (+) end Slower grow

8、ing (-) end cc = koff/konCc+Cc-Treadmilling occurs when Cc- Ccyto Cc+Actin filaments undergo treadmTreadmilling depends on the cytosolic concentration of ATP-bound G-actin14cc = koff/konW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. The cytosolic concentration of available su

9、bunits (= ccyto) determines whether a filament grows or shrinks. Treadmilling depends on the cyWhy do cytoskeletal filaments treadmill? W2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 15During treadmilling, the cell maintains the same overall filament content (while individual

10、 subunits cycle between filament and cytosol Is this a waste of energy?Treadmilling provides the cell with spatial and temporal flexibilityWhy do cytoskeletal filaments The cytosolic concentration of G-actin is tightly regulatedW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. T

11、he cytosolic concentration ofCapping proteins also regulate actin dynamicsCapping proteinsbind (+) or (-) endsfilament end is protected from depolymerization even if the available cytosolic monomer concentration (ccyto) is below the critical concentration of that particular end.W2011 D103 - lecture

12、5 Suetterlin and MacGregor - all rights reserved. Capping proteins also regulateExamples for F-actin capping proteinsW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 18The muscle sarcomere remains at a steady length.During a contraction, myosins and actin filaments slide agains

13、t each other Actin filaments in the muscle sarcomere do not treadmill.Examples for F-actin capping pVisualizing cytoskeletal components by immunofluorescenceW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 19Visualizing cytoskeletal compoHow to detect intracellular structures b

14、y immunofluorescenceW2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 20Concept: Cellular structures (organelles, cytoskeleton) are characterized by the presence of specific marker proteins (eg -tubulin for microtubules). These marker proteins can be used as antigens to raise sp

15、ecific antibodies. Antibodies to a specific antigen will therefore label the cellular structure in which the antigen is present and reveal its localization and organization inside a cell. Example: an antibody to a Golgi protein will reveal the organization of the Golgi apparatusAntibody to Mannosida

16、se II, a glucosyltransferase that is known to function in the Golgi apparatusHow to detect intracellular stHow is immunofluorescence done in a lab?W2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 21Fluorescent lightexcitationemissionLightNucleusmicrotubulesGolgiPrimary antibody

17、(Rabbit anti tubulin Ab)recognizes antigen (eg tubulin)Secondary antibody(Goat anti-rabbit Ab)recognizes the constant region of rabbit antibodiesRecognizes Golgi proteinFluorescent lightLightHow is immunofluorescence done more than one secondary antibody binds to one primary antibody signal enhancem

18、ent antibody coupling is inefficient - precious primary antibody would be lost during the coupling process. flexibility in the the color of the fluorochrome - combinatorial stainings with Green or Red fluorescent proteinsWhy are secondary antibodies used?W2011 D103 - lecture 5 Suetterlin and MacGreg

19、or - all rights reserved. 22 more than one secondary antibImportant concepts, lecture 5W2011 D103 - lecture 5 Suetterlin and MacGregor - all rights reserved. 23The cytoskeleton gives a cell mechanical support, but is dynamic.3 main cytoskeletal structures: - actin filaments and microtubules assemble into polar structures in an energy-dependent- intermediate filaments