Modulation of light-induced homo-

oligomerization of Cryptochrome2

Victor Acero1 , Liting Duan2, Jen Hope2 Bianxiao

Cui2

Pennsylvania State University1, Stanford University2

 Why do Protein-Protein Interactions
Matter?
Respiration Gene
Expression

Organelle Signaling
Function Pathways

2
Light Sensitive Proteins as a Tool to
Control Protein-Protein Interactions

CRY
2

DAR LIGH
K T
CIBN
CRY CRY CRY CRY CRY
2 2 2 2 CRY 2
2

3
How does CRY2/CIBN Help Study
Protein-Protein Interactions?

4
 Homo-Oligomerization as a Tool

OFF CRY
2
LIGH
T
ON
CRY CRY CRY CRY
2 2 2 2
CRY DAR
2
K

5
 Hetero-dimerization as a Tool

CIBN

OFF ON
CIBN
LIGH CRY
T 2

CRY
2 DAR
K

6
Improving the CRY2 tool by increasing
control
Modulation of
Homo-
Oligomerization

Increase Decrease
Oligomerization Oligomerization

Clustering Tool Noise Reduction

7
Modulation of CRY2 Homo-
Oligomerization via construct
engineering
CIBN

CRY
2

8
Research Question 1: What modulating effect do
fluorescent tags have on homo-oligomerization?

9
Modulation of Oligomerization on ER
Membrane via Fluorescent Tag

CRY mC Sec
2 h 61

LIGH
T
G
CRY F Sec
2 P 61

10
 Modulation of Oligomerization on
Cell Membrane via Fluorescent Tag

CRY mC
2 h CAAX

LIGH
T

G
CRY F
P CAAX
2

11
Fluorescent tags can be used to
increase or decrease homo-
oligomerization

>
Homo-
oligomerization 12
Research Question 2: Can we engineer
a platform using bulky-proteins to
modulate homo-oligomerization?

13
Previous Research Indicates Bulky
Proteins can Modulate Homo-
Oligomerization

Figure 5 (Daphne L. Che et al, 2015) 14

 Confirming Steric Hindrance of
Oligomerization using tdTomato
LIGH
tdTomato CIBN
T

CIBN G tdTomato
CRY F Sec LIGH
P
2 61 T

15
Conclusion: Fluorescent Proteins and Bulky
Proteins can Modulate CRY2 Homo-Oligomerization

Modulation of
Homo-
Oligomerization

mCher Increase Decrease GFP

ry Oligomerization Oligomerization Bulky
Proteins

Clustering Tool Noise Reduction

16
 Future Work
1. Fluorescent
Tags
2.
Modulate
Homo-
Bulky Oligomerizat
Proteins ion
Sterically
Hinder ???
Homo-
Oligomerizai
ton
Mechanisms
of
Oligomeriza
tion

17
Acknowledgements

18
Questions?

19
Editing Desired Constructs into
Plasmids
G
F Sec
CIBN
P 61

Ly
mC
n CRY
h
2

20
Cloning Plasmids via E coli

21
Transfection of Plasmids into COS7
Cells

Chemical
Transfection COS7
COS7

COS7
COS7 COS7
COS7

22
Quantification of Oligomerization:
Puncta Analysis

LIGH
T

23
 Previous Research: The Dual Characteristics of Light-Induced
Cryptochrome 2, Homo-
oligomerization and Heterodimerization, for Optogenetic
Manipulation in Mammalian Cells
Figure 5
(Daphne L.
Che et al,
2015)

Figure 5 (Daphne L. Che et al, 2015) 24

24
Previous Research Indicates Bulky
Proteins can Modulate Homo-
Oligomerization

Figure 5 (Daphne L. Che et al, 2015) 25