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RIC1-GFP Pollen
Morphology,
Growth patterns,
and Brefeldin A
Geoff Harlow & Mike Oliveira
3/15/12
Introduction
• Cell Polarity in Plants
• Pollen growth
• RIC1 and its role in the ROP1 signalling network
Why is cell polarity
important?
• Explains how cells make concerted efforts to
mechanically achieve survival

• Serves as ...
Examples of Cell Polarity &
Shape Formation in Plants
•
•
•
•

Pavement Cell
Root Hairs
Trichomes
Pollen Tubes

• Frontier...
Pavement Cells
Root Hairs
Trichomes
ROP1
GTP

ICR1 / RIP1

RIC3

ROP1
RIC4

GDP

SEC3

Ca2+

actin
disassembly

+

F-actin assembly

Polar exocytosis
Why was RIC1 selected?
• Interacts with ROP1 bound to GTP
o ROP1: Rho-GTPase of plants

• 224 amino acids
• Localizes to p...
Research Question
• Research Question:
o What is the effect of BFA on Pollen Tube Growth?

• Hypothesis:
o BFA will inhibi...
What is Brefeldin A?
• Fungal metabolite
• Blocks transport from ER  Golgi

Rough ER

Golgi

Nucleus
Vesicles
Cell Membra...
Methods
• (1) RIC1 fused with GFP
• (2) RIC1-GFP inserted into pollen genome
• (3)Pollen grown on slides containing agaros...
Image Analysis Methodology
Image
Image
Image
Image
Image
Image

Input: Video
Data

Threshold the
image

Remove noise

Find...
Image Analysis - Finding
Tube Region
Input Image

Threshold

Remove Noise

CC/Close Image
Gaps
Image Analysis - Finding
Tube Region
Input Image

Output Image
Image Analysis – Tip
Tracking
Input Image

Find
Symmetry/Tip

Define Search
Area

Final Image
Growth
• Input Data:
o Tube mask images

• Extract area in each image
• Normalize area to first image
• Plot Normalized Ar...
Tip Tracking – RIC1
Expression
• Input Data:
o Segmented tube images
o Segmented tip images

• Determine average intensity...
Results - Control
Results - 1uM BFA
Results - 5uM BFA
Results - 10uM BFA
• No growth detected, but interesting observations

What is this?
Z-Stack of 10uM BFA
Treated Pollen Grain (A)
Z-Stack of 10uM BFA
Treated Pollen Grain (B)
Results - Growth
1.2

Normalized Area

1.15

1.1

Control

1.05
1

1 uM BFA

0.95

5 uM BFA

0.9
0.85
0.8
0

50

100

150
...
Results – Tip Intensity
Normalized Intensity

1.3
1.2
1.1
1

Control
1 uM BFA
5 uM
BFA

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0

50
...
Results – Growth and
RIC1 Expression
Control

1 uM BFA

1.2
1
0.8
0.6
0.4
0.2

1.4

Normalized Area/Intensity

1.4

Normal...
Discussion Questions
• Does the data support that RIC1 actually oscillates?
• Are there lag and go phases of total pollen ...
Conclusions
• RIC1 appears to oscillate, but it’s not definitive
• 1uM BFA
o RIC1 does not seem to localize at the membran...
Future Experiments
• Other drugs that inhibit/enhance exocytosis:
o Wortmannin
o Latrunquilin B
o Jasplakinolide

• Other ...
References
• Yuan Qin, Zhenbiao Yang. “Rapid tip growth:
Insights from pollen tubes.” Seminars in Cell &
Developmental Bio...
Appendix:
MATLAB Functions
Software Function
Read images
Threshold an image
Remove small clusters
Close region gaps
Find c...
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RIC1 GFP pollen morphology, growth patterns, and Brefeldin A

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Final presentation for EE 273: Live Imaging & Analysis of Cellular & Molecular Behavior

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RIC1 GFP pollen morphology, growth patterns, and Brefeldin A

  1. 1. RIC1-GFP Pollen Morphology, Growth patterns, and Brefeldin A Geoff Harlow & Mike Oliveira 3/15/12
  2. 2. Introduction • Cell Polarity in Plants • Pollen growth • RIC1 and its role in the ROP1 signalling network
  3. 3. Why is cell polarity important? • Explains how cells make concerted efforts to mechanically achieve survival • Serves as a model for mechanical construction that has stood the test of time (millions of years!)
  4. 4. Examples of Cell Polarity & Shape Formation in Plants • • • • Pavement Cell Root Hairs Trichomes Pollen Tubes • Frontiers of scientific inquiry revealed through imaging!
  5. 5. Pavement Cells
  6. 6. Root Hairs
  7. 7. Trichomes
  8. 8. ROP1 GTP ICR1 / RIP1 RIC3 ROP1 RIC4 GDP SEC3 Ca2+ actin disassembly + F-actin assembly Polar exocytosis
  9. 9. Why was RIC1 selected? • Interacts with ROP1 bound to GTP o ROP1: Rho-GTPase of plants • 224 amino acids • Localizes to plasma membrane • Contains CRIB domain o CRIB: cdc42/Rac-interactive binding • Serves as good reporter for tip growth
  10. 10. Research Question • Research Question: o What is the effect of BFA on Pollen Tube Growth? • Hypothesis: o BFA will inhibit pollen tube growth and RIC1 expression at the tip due to inhibition of exocytosis
  11. 11. What is Brefeldin A? • Fungal metabolite • Blocks transport from ER  Golgi Rough ER Golgi Nucleus Vesicles Cell Membrane
  12. 12. Methods • (1) RIC1 fused with GFP • (2) RIC1-GFP inserted into pollen genome • (3)Pollen grown on slides containing agarose gel o o o o o o 18% Sucrose 5% Boric Acid 1M MgSO4 1M Ca(NO3)2 1M CaCl2 +Brefeldin A @ 1uM, 5uM, 10uM • (4) Incubated 3hrs @ 30 degrees C. • (5) Visualized using Leica SP2 confocal microscope • (6) Analyzed using MATLAB
  13. 13. Image Analysis Methodology Image Image Image Image Image Image Input: Video Data Threshold the image Remove noise Find edges and create line of symmetry Morphological image closing to create solid region Find tip and define search region TUBE AREA TUBE INTENSITY Connected Components TIP INTENSITY Growth – Normalized Area (Arean / Area1) Tip Intensity – Normalized Intensity ( IntTIP / IntTUBE )
  14. 14. Image Analysis - Finding Tube Region Input Image Threshold Remove Noise CC/Close Image Gaps
  15. 15. Image Analysis - Finding Tube Region Input Image Output Image
  16. 16. Image Analysis – Tip Tracking Input Image Find Symmetry/Tip Define Search Area Final Image
  17. 17. Growth • Input Data: o Tube mask images • Extract area in each image • Normalize area to first image • Plot Normalized Area vs. Time
  18. 18. Tip Tracking – RIC1 Expression • Input Data: o Segmented tube images o Segmented tip images • Determine average intensity o Total pollen tube o Pollen tube tip • Normalize intensity o Tip / Tube • Plot Normalized Intensity vs. Time
  19. 19. Results - Control
  20. 20. Results - 1uM BFA
  21. 21. Results - 5uM BFA
  22. 22. Results - 10uM BFA • No growth detected, but interesting observations What is this?
  23. 23. Z-Stack of 10uM BFA Treated Pollen Grain (A)
  24. 24. Z-Stack of 10uM BFA Treated Pollen Grain (B)
  25. 25. Results - Growth 1.2 Normalized Area 1.15 1.1 Control 1.05 1 1 uM BFA 0.95 5 uM BFA 0.9 0.85 0.8 0 50 100 150 200 Time (s) 250 300 350
  26. 26. Results – Tip Intensity Normalized Intensity 1.3 1.2 1.1 1 Control 1 uM BFA 5 uM BFA 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0 50 100 150 200 Time (s) 250 300 350
  27. 27. Results – Growth and RIC1 Expression Control 1 uM BFA 1.2 1 0.8 0.6 0.4 0.2 1.4 Normalized Area/Intensity 1.4 Normalized Area/Intensity Normalized Area/Intensity 1.4 5 uM BFA 1.2 1 0.8 0.6 0.4 100 200 300 400 Time (s) Normalized Area Normalized Intensity 1 0.8 0.6 0.4 0.2 0.2 0 1.2 0 100 200 Time (s) 300 400 0 100 200 Time (s) 300 400
  28. 28. Discussion Questions • Does the data support that RIC1 actually oscillates? • Are there lag and go phases of total pollen growth? • What is the “cashew” shape inside of the pollen grains?
  29. 29. Conclusions • RIC1 appears to oscillate, but it’s not definitive • 1uM BFA o RIC1 does not seem to localize at the membrane • 5uM BFA o Appears to inhibit growth o RIC1 does not seem to localize at the membrane • 10uM BFA o appears to inhibit growth o Produces “cashew” shaped RIC1 aggregates centrally
  30. 30. Future Experiments • Other drugs that inhibit/enhance exocytosis: o Wortmannin o Latrunquilin B o Jasplakinolide • Other potential genes to label: o o o o SEC3 ICR1 RIP1 PMEi • Optimizing growth conditions: o Reduce time between incubation interruption and visualization
  31. 31. References • Yuan Qin, Zhenbiao Yang. “Rapid tip growth: Insights from pollen tubes.” Seminars in Cell & Developmental Biology, Volume 22, Issue 8, October 2011, Pages 816-824 • Yan Zhang, Junmin He, David Lee, and Sheila McCormick. “Interdependence of Endomembrance Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes.” Plant Physiol. Vol. 152, 2010, Pages 22002210 • http://arabidopsis.org/servlets/TairObject?id=34472 &type=gene
  32. 32. Appendix: MATLAB Functions Software Function Read images Threshold an image Remove small clusters Close region gaps Find connected components MATLAB Function imread() graythresh() bwareaopen() imclose() bwconncomp()

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