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Research Interests

We are interested in understanding how cytoskeleton influences cellular morphogenetic processes during normal development or after injury. We use a variety of techniques, including stem cell biology, molecular biology, biochemistry, biophysics, and microscopy to study morphogenesis in neurons.

 

Acentrosomal Microtubule Nucleation in Neurons

Neuronal morphogenesis is the process through which a nerve cell (neuron) generate its elaborated axon and dendrites. It is the fundamental process underlying the establishment and plasticity of neuronal networks. Neuronal morphogenesis relies on the a fiber-like scaffold protein complex called microtubule cytoskeleton. It is well established that nearly all microtubules inside a mitotic cell originate from a microtubule-organizing center (MTOC) called centrosome. Interestingly, the centrosome in neurons does not seem to act as the MTOC. We recently discovered a microtubule-associated protein called TPX2 plays an important role in acentrosomal MTOC. We previously showed that microtubule-associating protein kinase A (PKA) influence neuronal morphogenesis (the original paper can be found here). The image below shows an isolated hippocampal neurons undergoing neuritogenesis. This hippocampal neuron was fixed and stained for microtubule (red), F-actin (green), and the nucleus (blue).

mHN

Axon Regeneration

The other interest of the lab is the axon regeneration process, it happens after extended axons are severed. The central nervous system (CNS) in mammals is incapable of regeneration while the peripheral nervous system (PNS) shows various degrees of regeneration. We are interested in studying the mechanistic basis of this CNS-PNS difference in axon regeneration. Additionally, we are developing novel methods (e.g. surface topology or nanoparticles) to accelerate axon regeneration. The movie below shows rat DRG axons undergoing robust axon regeneration after axotomy.

DRG_reg

 

 

Optical Manipulation of Cells

In order to study the role of specific cytoskeleton or organelles in neuronal morphogenesis and axon regeneration, we are utilizing a variety of optical manipulation methods. For example, we have constructed a laser optical tweezers system to position micrometer-sized cellular structure or to change cell shape in living cells or neurons (see image below). In addition, we are developing photoactivatable or photoinactivatable proteins to study the function of proteins in a spatial- and temporal-specific manner.

LOT new

 

 

High-content Drug or RNAi Screening

In addition, we are using high-content screening technology to look for drugs, RNAi molecules (siRNA, shRNA, or miRNA), or proteins that can influence axon regeneration or neuritogensis. High-content screen is an microscopy-based high-throughput screen, which takes advantage of the automated image acquisition machinery and image analysis software to screen large volume of drugs or biomolecules within a short amount of time. By combining primary neuronal culture and lentivirus-mediated transduction, we can efficiently suppress endogenous protein in a physiologically relevant model system.

HCS

The images below show primary hippocampal neurons under two different treatments. Virus infected neurons appear in green (GFP expression), the "protein of interest" is shown in red, and a neuron-specific marker is shown in blue. The neurons in the right (or bottom) image have their protein of interest suppressed and result in shorter neurites.

HCS1 HCS2

 

 

High-throughput Quantification and Classification of Neurons

In the bioinformatics section of the lab, we are developing high-throughput image analysis methods for quantifying and classifying neurons based on their morphology. Due to the advancement of image acquistition machinery, large volume of data can be generated over a short period of time. How to accurately and efficiently extract morphological features from these images has become a challenge for modern cell biology lab. We are collaborating with our bioinformatics colleagues to develop image analysis algorithms which can automatically detect neuronal morphological features (our freely available software can be download from here).

HCA analysis flow

 

 

Eric Hwang, Associate Professor
Department of Biological Science and Technology
Room 111 Zhu-Ming Building, 75 Bo-Ai Street
Hsinchu, Taiwan 300
Tel: +886-3-5712121#56968
Fax: +886-3-572-9288
hwangeric (at) mail.nctu.edu.tw

All rights reserved. © 2017 Eric Hwang