Elektronmikroskopi för att titta in i celler och mikroorganismers nano

Elektronmikroskopi för att titta in i celler och mikroorganismers nano-universum. •  Eukaryota cellers cytoskelett •  Bakteriers cytoskelett •  Elektronmikroskopi

Linda Sandblad Institutionen för Molekylärbiologi 2015-10-27

Nano-universums skala

Cytoskelettet har en central roll i cellernas funktion. Skapar cellens infrastruktur och rumsliga organisation. Interagerar med olika typer av cytoskelettbindande proteiner.

https://www.youtube.com/watch?v=FzcTgrxMzZk

•  SEM = Scanning Electron Microscop •  TEM = Transmission Electron Microscop

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Ljus mikroskop

Figure 9-42 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

TEM

Transmission Electron Microscopy

TEM

Bacteria

Proteins in solution

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SEM

Scanning Electron Microscope (Svepelektronmikroskop)

Figure 9-49 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

By SEM

File:Miridae SEM 3.jpg

Cytoskelettet är polymerer av protein Protein sätts ihop till filament - Polymeriserar – långa trådar eller stavar i alla celler

Direct visualization of secondary structures of F-actin by electron cryomicroscopy Takashi Fujii,1 Nature October 2010

Actin

Microtubules

EB1-GFP

αβ-Tubulin + Taxol assemble into microtubule in vitro β

+

+GTP

Taxol

α Nogales et al., 1998

-

Dynamic Instability is driven by GTP hydrolysis

Figure 16-16a Molecular Biology of the Cell (© Garland Science 2008)

Sample preparation for EM – Negative staining

Protein in solution

Heavy metal salt

What you see in the TEM

= Freezing a hydrated solution without crystallization •  Rapid freezing turns water in to a amorphous solid state •  Sample have to stay below -140° C •  Vitrifies samples are transparent for a electron beam Negative staining

Frozen hydrated by plunge freezing Cryo-EM

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The key is RAPID cooling

Starts at -140°C to -180°C 18

Duboche and Fuller

Sample preparation for cryo-EM

Plunge freezing

Grid

Nitrogen Ethane

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Vitrified sample in the carbon holes

20-200 nm

gold

sample

carbon

Linda Sandblad

1-4 µm

Vitrified buffer/water

Fråga: The electron micrograph shown in figure A were obtained from a population of microtubules that were growing rapidly. Figure B was obtained from microtubules undergoing “catastrophic” shrinking. Comment on any differences between A and B, and suggest likely explanations for the differences that you observe.

The bacterial cytoskeleton.

Matthew T. Cabeen, and Christine Jacobs-Wagner J Cell Biol 2007;179:381-387

•  •  • 

Tubulin Actin Intermediate filaments

= = =

FtsZ - Cell division MreB, ParM - DNA segregation Crescentin, FilP - Cell shape

FtsZ-GFP

FtsZ negative staining EM

FtsZ ring model

The actin like cytoskeleton

Figure 16-27a Molecular Biology of the Cell (© Garland Science 2008)

Homology between prokaryotic and eukaryotic cytoskeletal filaments.

Bill Wickstead, and Keith Gull J Cell Biol 2011;194:513-525

© 2011 Wickstead and Gull

Crescentin, a intermediate filament in Caulobacter crescentus

Ausmees et al. 2003

Electron Tomography

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