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
4
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
7
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
17
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
19
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
27
