Yeast Actin Filaments:

1

• Yeast Actin Filaments
• Effects of Site-Directed Mutation and Cysteine
  Labeling on Filament Structure

2
Actin and Muscle

• Filaments and Contraction

3
This is a short section of the thin filaments
helix. Each colored area represents one actin
monomer. I created this image using InsightII, a
3-D molecular modeling program.
4
Rabbit vs. Yeast Actin
Rabbit G-actin
Yeast G-actin
Cysteines at residues 17, 217, 285, and 374.
Cysteines at residues 10, 217, 257, and 285.
Structurally Similar But which is really which?
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Helices, Sheets Cysteines

• Key structural elements shown.
• Helices are orange.
• Sheets are red.
• The ribbon structure is purple.
• The cysteines are fully rendered.

6
The Muscle Cycle
7
Transmission Electron Microscopy

• On a Jeol 1200-EX TEM

8
Electron Microscope Grids
A film-less copper grid
My finger

• A typical grid has 300 grid squares.
• Grids are quite fragile, and bend or break
  easily.
• At 30,000x to 40,000x magnification, that
  necessary to see actin filaments, a small area of
  a single grid square is seen.

9
The Jeol 1200-EX
Lens Controls
Mini-screen
Coolant Tank
Screen Toggle
Binoculars
Main Screen
Sample Rod
Chamber
X-Axis Scroll Wheel
Column
Y-Axis Scroll Wheel
10
(Not to Scale)
Filament
Magnetic Lens (Acceleration)
Electron Beam
Sample Actin Filament
Magnetic Lens (Focus, Magnification)
Reflected Radiation
Phosphorescent Screen
Image of Actin Filament
Photographic Film
Exposed Film Image
11
Site-Directed Spin Labeling

• Spin Label
• Observed using EPR.
• Electron spin states in this molecule absorb
  microwave radiation when in a strong magnetic
  field.

• Maleimide
• Reacts well with cysteines.

Why? These spin labels, when attached to
cysteines near acto-myosin interaction sites on
the actin monomer, could reveal more information
about the movement of both thick and thin
filaments in muscle.
12
Methods
13
QuikChange

• Creating the Mutants

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Mix and Cycle the Solutions
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Digest Parental DNA and Transform Plasmid
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Plasmid Repair

• The E. Coli cells accept the new DNA.
• Their cellular processes repair any damage done
  to the plasmid, as well as completing the mutated
  sequences insertion into the DNA by removing any
  nicks.

17
Extracting the Yeast Actin
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DNase Column

• Lyse yeast cells in a bead beater
• DNase column binds proteins and genetic material
• Create a DEAE DE52 resin
• Using these solutions, wash out unwanted junk
• G-B/PI, G-B/NH4Cl, and 1X G-Buffer
• Elute the actin into a DEAE DE52 column with
  G-B/50 Formamide
• Elute the actin with G-B/KCl
• Place the actin in dialysis against G-B/PI/DTT

19
Preparing TEM Grids

• Determine actin concentration using
  spectrophotometer
• Dilute actin to 1 mg/mL keep on ice
• Polymerize by adding KCl to solution
• Dilute small samples to desired concentrations
• Pipette one drop of actin onto TEM grid
• Wick excess solution away with filter paper
• Pipette one drop of UAc at 1 g/100 mL on top of
  actin on grid
• Wick away excess allow to dry

20
Results

• Electron Micrographs

21

• Rabbit actin,
• 0.1 mg/mL, formvar grid
• Preliminary observations.
• Good filament density for observations of
  structure.
• This concentration will be good for observing
  yeast actin.

22

• Wild-type yeast actin, unlabelled,
• 0.1 mg/mL, formvar grid
• Less filament density than rabbit actin.
• Implies lower stability.
• Long, unmodified filaments are stable and not
  clumped.

23

• Wild-type yeast actin, unlabelled,
• 0.1 mg/mL, carbon-formvar grid
• Very sparse filaments.
• Implies much lower stability.
• Only a few long filaments visible.
• Lots of short fragments.
• Grid type affects stability?

24

• Wild-type yeast actin, labelled, 0.25 mg/mL,
  carbon-formvar grid
• Dense, clumped filaments.
• Change in inter-filament bonding?
• Pock-marks in the stain near filaments.
• Filaments are still full length.
• Labeling affects bonding?

25

• Wild-type yeast actin, labelled,
• 0.25 mg/mL, carbon-formvar grid
• Stringy, clumped filaments.
• Change in inter-filament bonding?
• Filaments are still full length.
• Labeling affects bonding?

26

• Wild-type yeast actin, labelled, 0.25 mg/mL,
  carbon grid
• Short, clumped filaments.
• Change in inter-filament bonding?
• Pock-marks in the stain near filaments.
• Labeling combined with a different grid-type
  affects bonding and stability?

27

• Wild-type yeast actin, labelled,
• 0.5 mg/mL, carbon-formvar grid
• Short, clumped filaments.
• Change in inter-filament bonding?
• Pock-marks in the stain near filaments.
• Longer filaments
• Labeling affects bonding?
• Increased concentration increases filament
  stability?

28

• Wild-type yeast actin, labelled,
• 0.5 mg/mL, carbon grid
• Long, densely clumped filaments.
• Significant change in inter-filament bonding?
• Densely pock-marked near filaments.
• Labeling causes filaments to change their bonding?

29

• M1C mutant yeast actin, unlabelled,
• 0.5 mg/mL, carbon-formvar grid
• No visible filaments.
• High instability caused depolymerization?
• Large pock-marks throught the stain.
• M1C mutation prevents monomers from polymerizing
  correctly?

30
Credits

• Dr. Vicci L. Korman - Supervisor
• Dr. David D. Thomas - Outside Advisor
• Chelen H. Johnson - Teacher
• Dr. Jacob Miller - Summer Teacher
• Thanks also to others at the Thomas Lab, and to
  the EM people for letting me use their scope.

31

• Yeast Actin Filaments
• Effects of Site-Directed Mutation and Cysteine
  Labeling on Filament Structure