Jordan Gottlieb’s Research at the

Goode Lab Summer 2009 Science Internship

Overall Director: Dr. Bruce Goode – Principal Investigator Mentor: Erin Jonasson – Ph.D. Student, Molecular/Cell Biology

Location: Brandeis Univeristy Rosenstiel Center MS 029415 South StreetWaltham, MA 02454

GFP tagActin Cytoskeleton

Arp 2/3 complex

The Lab

• The Goode lab itself focuses on how actin filaments are manipulated and regulated in budding Yeast Cells.

• My particular research focused on the protein Hof1 involving its role in formin-mediated actin inhibition.

KDa

Purified Hof 1

Hof1-GFP Bnr1-GFP

Hof1 belongs to the PCH (Pombe Cdc15 Homology) family proteins, which regulate membrane & actin dynamics

Chitu & Stanley, 2007. Trends Cell Biol. 17: 145-56.

CCFCH SH31 669Hof1Yeast

Human

Hof1 belongs to the PCH (Pombe Cdc15 Homology) family proteins, which regulate membrane & actin dynamics

Membrane Actin CytoskeletonRegulation

F-Bar SH31 340 603 670

Hof1

WASp-Arp2/3 Actin Assemblyendocytosis

Formin Actin AssemblyCytokinetic Acitn Ring

Polarized Cables

Hof1 inhibits Bnr1(SH3)

Concentration of Hof1 construct (nM)0 5 10 15 20

40

60

80

100

20

0

GST-SH3

Hof1∆SH3

FL Hof1

Kapp~ 93nM

Kapp~ 4nM

Kapp~ 1.5nM

% B

nr1(

FH2)

act

ivit

y

F-Bar SH31 340 603 670

Hof1

F-BarHof1∆SH3

SH3SH3

Inhibition Activity

++++

+++

+

0 50 100 150 200 250 300

100

80

60

40

20

0

GST-SH3 Kapp ~ 93nM%

Bnr

1(FH

2) a

ctiv

ity

[GST-SH3] (nM)

Elsie’s and Felicia’s Hof 1 Constructs

What is the other inhibitory domain of Hof1?

F-Bar SH31 340 603 670

Hof1

F-BarHof1∆SH3

SH3SH3

++++

+++

+

Inhibition Activity

++++

+++

+

FH2Binding

F-BarF-Bar

SH3Hof1∆BAR

Hof1∆BAR∆SH3

What Erin and I set out to create this Summer:

My Work Station

My Commassie Protein Gel of the Hof 1 (F-bar only) construct

KDa

66

1 2 3 4

F-bar domain Hof 1 construct at 64 KDa

Lane 2 Uninduced Control in BL21competent cells

Lane 3Induced TestIn BL21 competent cells

Lane 4Uninduced ControlIn Rosettacompetent cells

Lane 5 Induced TestIn Rosetta competent cells

200

94

45

31

Acknowledgements

Dr. GoodeGoode Lab MembersErin

Elsie Felicia

Sasha BearSpot

Michele