Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons
Summary
The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software “KYMOMAKER” are shown here.
Abstract
Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software “KYMOMAKER” is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.
Introduction
Kinesin family members and cytoplasmic dynein are motor proteins that move along the microtubules in cells to transport their cargo1. Most kinesins move toward the plus end, whereas dynein moves toward the minus end of a microtubule. The functions and mechanisms of cargo transport in the neuronal axon have been investigated extensively. Because of their length, axons require stable long-distance transport to maintain the health of neurons. Defects in the transport of mitochondria, autophagosomes, and vesicles containing amyloid-β protein precursor (APP) have been reported as causes of neurodegenerative diseases2,3. Numerous in vitro investigations have revealed the mechanisms underlying coordinated transport by motor proteins, and various studies using purified motor proteins and microtubules have uncovered how motor molecules move along microtubules4,5. Typically, multiple motors are involved in a single cargo6. However, there are some models of how the opposite motors determine the direction of cargo transport. One is the "association/dissociation model"; in this, only one-directional motors are associated with the cargo during the one-directional transport. In the second, the "coordination model", both motors are attached to the same cargo, and only one side of the motor is activated. In the third "tug-of-war model", the force balance between kinesins and dyneins determines the direction of transport7,8,9. In addition, several reports have suggested that the balance and number of activated motor proteins influence the velocity of cargo transport in vitro8,10.
An unanswered question is how these activities of kinesins or dyneins are regulated in living cells. Previous reports have shown acetylation of microtubules in axons, and neurotrophic factors enhance axonal transport11,12. Also, various types of cargo and cargo adapters function as motor activators in corresponding ways13,14. Many are associated with the membrane of transport vesicles, and their functions are regulated by signals such as post-translational modifications15. Therefore, observing transport direction and velocity in living cells offers valuable insight into the molecular regulation of cargo transport in in vitro experiments. The observation of axonal transport allows distinguishing between kinesin- and dynein-based transport. Because axons harbor plus-end unidirectional microtubules16, cargo is transported anterogradely (i.e., soma to axon terminal) by kinesins and retrogradely (i.e., axon terminal to soma) by dyneins.
In the present study, a method to observe and analyze axonal transport in primary cultured neurons is described. As an example, the procedure for observing the axonal transport of membrane proteins-APP, calsyntenin-1/alcadein α (Alcα), and calsyntenin-3/alcadein β (Alcβ)-containing vesicles-is described. It is known that the anterograde transport of APP-containing vesicles is considerably faster than that of Alcα-containing vesicles, although both are transported by kinesin-117,18,19. In previous reports, several methods have been used to measure velocity. The most variable step is the handling of pauses during transport. In live cells, transport is sometimes hindered by obstacles along the microtubules; however, motors can bypass a region with or without a pause20. The calculation of velocity over a longer observation time may be affected by the pause, which can result in a slower speed estimation. Here, a method is described using movement over a segmented (200 ms) period to exclude the effect of the physical pause of motors. Finally, an open-access (Windows only) software program called "KYMOMAKER"21 is introduced. Kymographs are used widely to visualize vesicular transport and are useful for visualizing the transport direction of each cargo without requiring a movie. The software generates kymographs from time-lapse movies by applying a one-dimensional Watershed algorithm multiple times during the rotation of images. This enables the resulting kymograph to show fine structures efficiently and easily. Furthermore, KYMOMAKER automatically detects and highlights trails according to their direction and enables the creation of easy-to-understand diagrams.
Protocol
Representative Results
Discussion
An analysis method for axonal transport is described, which includes the calculation of segmented velocity and the generation of kymographs. A critical step during the transfection step is maintaining the health of cultured neurons. The transfection method described by Jiang and Chen29 was followed with minor modifications. Gentle mixing of the DNA/CaCl2 solution and 2x HBS increased the efficiency of transfection by reducing the size of the precipitations. To prevent the precipitations…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by KAKENHI (22K15270, Grant-in-Aid for Young Scientists) and the Akiyama Life Science Foundation for YS. We would like to express our gratitude to Dr. Masataka Kinjo and Dr. Akira Kitamura, Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University for providing critical input and expertise that greatly assisted the research. The observation with TIRF microscopy was performed using the instrument installed at the Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University. The Instrument is registered in the Open Facility system managed by the Global Facility Center, Creative Research Institution, Hokkaido University (AP-100138). We thank Dr. Seiichi Uchida, Human Interface Laboratory, Department of Advanced Information Technology, Faculty of Information Sciences and Electrical Engineering, Kyushu University, Fukuoka Japan, for the assembly with the application Kymomaker. Advanced Prevention and Research Laboratory for Dementia, Graduate School of Pharmaceutical Sciences, Hokkaido University is supported by Japan Medical Leaf co., Ltd.
Materials
| 5-Fluoro-2-deoxyuridine | Sigma-Aldrich | F0503 | |
| Apo TIRF 100x/1.49 OIL | Nikon | ||
| B-27 Supplement (50x), serum free | Thermo fischer scientific | 17504044 | |
| Bovine serum albumin | Wako | 013-25773 | |
| CalPhos Mammalian Transfection Kit | Takara | 631312 | |
| Cell strainer 40 µm Nylon | Falcon | 352340 | |
| CoolSNAP HQ | Photometrics | ||
| Deoxyribonuclease I | Sigma-Aldrich | DN-25 | |
| D-Glucose | Wako | 041-00595 | |
| DMEM/Ham’s F-12 | Wako | 042-30555 | |
| Dumont No. 7 forceps | Dumont | No.7 | |
| Feather surgical blade | Feather | No.11 | |
| Feather surgical blade handle | Feather | No. 3 | |
| Gentamicin | Wako | 079-02973 | |
| Gentamicin Sulfate | Wako | 075-04913 | |
| GlutaMAX Supplement | Thermo fischer scientific | 35050061 | |
| HEPES | DOJINDO | 342-01375 | |
| Horse Serum, heat inactivated | Thermo fischer scientific | 26050088 | |
| KCl | Wako | 163-03545 | |
| KH2PO4 | Wako | 169-04245 | |
| KYMOMAKER | http://www.pharm.hokudai.ac.jp/shinkei/Kymomaker.html | ||
| L-15 Medium (Leibovitz) | Sigma-Aldrich | L5520 | |
| MetaMorph version 6.2r1 | Metamorph | ||
| Na2HPO4 | Wako | 197-02865 | |
| NaCl | Wako | 197-01667 | |
| NaHCO3 | Wako | 191-01305 | |
| Neurobasal Medium | Thermo fischer scientific | 21103049 | |
| Nikon ECLIPSE TE 2000-E | Nikon | ||
| Nunc Lab-Tek 8 well Chambered Coverglass | Thermo fischer scientific | 155411 | |
| Papain | Worthington | LS003126 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | |
| Poly-L-lysine hydrobromide | Sigma-Aldrich | P2636-500MG | |
| Trizma base | Merck | T1194-10PAK | solved with water to make 0.1 M Tris-HCl (pH.8.5) |
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