Cortical microtubule labeling: insight of AFH14 in non-dividing cells
Chao Cai1, Yanhua Li, Yuan Shen
1Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education and College of Life Science, Beijing Normal University, Beijing, People's Republic of China.
Abstract:
We recently reported that AFH14 participated in microtubule and actin filament interaction in cell division, and the AFH14 (FH1FH2) was important to the directly binding activity of microtubules and microfilaments. To preliminarily understand the function and localization of AFH14 in non-dividing cells, we overexpressed FH1FH2-RFP in onion epidermal cells, and found a fluorescence labeled filamentous network. The result of double labeling with different cytoskeleton reporter proteins indicated that FH1FH2-RFP co-localized with cortical microtubules. Treatment of cells expressing FH1FH2-RFP with cytoskeleton disrupting drugs confirms that FH1FH2-RFP binds to microtubules. Moreover, the binding of FH1FH2-RFP to microtubules were revealed to be dynamic by fluorescence recovery after photobleaching (FRAP) experiment. Time-lapse confocal microscopy showed that FH1FH2-RFP could display a dynamics similar to the microtubule dynamic instability. These data suggest that FH1FH2 domain may lead AFH14 function on cortical microtubules in non-dividing cells, and FH1FH2-RFP may be utilized as a microtubule reporter protein in living onion epidermal cells.
Insights
The AFH14 FH1FH2 domain binds to microtubules in non-dividing onion cells. This domain exhibits dynamic behavior, similar to microtubule instability, suggesting a role in microtubule function.
Area of Science:
- Cell Biology
- Cytoskeleton Dynamics
- Plant Cell Biology
Background:
- AFH14 protein's role in microtubule and actin filament interaction during cell division was previously established.
- The FH1FH2 domain of AFH14 is crucial for direct binding to microtubules and microfilaments.
Purpose of the Study:
- To investigate the function and localization of AFH14 in non-dividing cells.
- To characterize the behavior of the AFH14 FH1FH2 domain in living plant cells.
Main Methods:
- Overexpression of FH1FH2-RFP fusion protein in onion epidermal cells.
- Double labeling with cytoskeleton reporter proteins.
- Treatment with cytoskeleton-disrupting drugs.
- Fluorescence Recovery After Photobleaching (FRAP) experiments.
- Time-lapse confocal microscopy.
Main Results:
- FH1FH2-RFP formed a filamentous network co-localizing with cortical microtubules.
- FH1FH2-RFP binding to microtubules was confirmed by drug treatments.
- FRAP revealed dynamic binding of FH1FH2-RFP to microtubules.
- The dynamics of FH1FH2-RFP mimicked microtubule dynamic instability.
Conclusions:
- The FH1FH2 domain likely mediates AFH14's function on cortical microtubules in non-dividing cells.
- FH1FH2-RFP serves as a potential reporter protein for studying microtubules in living onion epidermal cells.
