Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.
Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (“map”) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. “Cargo mapping” consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to “map” them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.
Intracellular transport is critical in all cell types for the delivery of proteins, membranes, organelles, and signaling molecules to various cellular domains1. Neurons are highly specialized cells with long, polarized projections that critically depend on intracellular transport of essential cargoes for their long-distance delivery to various axonal microdomains. This transport is mediated by kinesins and dyneins — two large families of molecular motor proteins — that bind to cargoes and track along polarized microtubules in anterograde and retrograde directions, respectively. While retrograde movement is mainly mediated by dynein, movement in the anterograde direction is facilitated by a large, functionally diverse family of kinesin motors. Consequently, anterograde transport of axonal cargoes could be mediated by various family members of the kinesin superfamily1-5. Though some cargoes move persistently in either direction, most cargoes move bidirectionally and reverse frequently on their way to their final destinations1,5-13. Furthermore, it has been shown that motors of opposing directionality associate simultaneously to cargoes, raising the question as to how regulated movement of cargoes is coordinated by opposite-polarity motors5-7. Together, transport of axonal cargoes is a concerted process that is regulated by the composition of motors and their specific biochemical activities, which in turn are dependent on various adaptors and regulatory binding partners14.
To faithfully describe the mechanism of axonal transport for a specific cargo and to uncover the underlying regulation of that transport, it is paramount to determine the composition of motor proteins and their regulatory binding partners associated with individual cargoes during their live transport. Other methods, for example biochemical approaches, provide estimates of average motor compositions on purified heterogeneous vesicle populations, but these estimates do not reveal the type or amounts of motors associated to single moving vesicles. Also, reconstitution of vesicle transport along preassembled microtubules in vitro enabled measuring the amount of one type of motor on a single vesicle level15. However, these experiments did not directly correlate the amount of motors with the transport characteristics of those vesicles, and measured transport in the absence of cellular regulatory factors.
A protocol is presented here, which determines the motor composition (type and relative amount of motors) of individual moving vesicles from immunofluorescence (IF) data measuring endogenously expressed motor proteins, and correlates these parameters to the live transport of the exact same vesicles in neurons16. This method entails precise mapping of IF-to-live cargo movement data. This is accomplished by growing hippocampal mouse neurons in microfluidic devices following established protocols17-19. These devices allow for the identification and correlation (“mapping”) of axons and single moving cargoes in fixed and live light microscopy modalities (Figure 1). Cultured neurons are transfected with fluorescently labeled cargo proteins whose transport is imaged at high spatial and temporal resolution to obtain detailed movement information that is plotted in kymographs. During the course of imaging, neurons are fixed with paraformaldehyde, and subsequently stained with antibodies against endogenous motor proteins. Fixed cargo and motor images are superimposed onto live movement kymographs to “map” (colocalize) them to the live cargo movement trajectories16. To correlate the live movement of cargoes with the association of motor proteins, colocalization is analyzed using a custom made MATLAB software package called “Motor Colocalization”16,20. Fluorescently labeled cargoes and motors generate diffraction-limited punctate features that can partially overlap. To resolve the position of overlapping puncta, the software first automatically fits Gaussian functions to each point spread function, representing individual fluorescent puncta, to determine their precise X-Y sub-pixel position coordinates and intensity amplitudes21-23. The positions of motors and cargoes are subsequently compared to each other to determine colocalization16,20. Therefore, this method more precisely assigns colocalization between fluorescent puncta as compared to other methods24.
The strength of this method is the ability to assess the colocalization of motors with individual cargo in fixed cells, for which live movement trajectories (e.g., the direction in which they were moving at the time of fixation) have been recorded. With this method, kinesins and dyneins were found to associate simultaneously to vesicles that carry the normal prion protein (PrPC-cellular), a neuronally enriched cargo that moves bidirectionally or remains stationary in axons16. This analysis allowed the formulation of a working model for the regulation of PrPC vesicle movement in which anterograde (kinesin) and retrograde (dynein) motors coordinate their activities in order to move the vesicles in either direction or to remain stationary while associated to the cargo. Another strength of this method is its potential broad applicability for characterizing colocalization/association of many fluorescently labeled cargoes that move in virtually any cell type, with any other protein(s) of interest. Thus, live/fixed correlation could potentially allow for the detection of transient protein-cargo interactions, as many individual fluorescently labeled moving particles can be analyzed over a desired period of time. Given the broad applicability and the type of questions that this method can address, this protocol will be of interest to a wide audience of cell biologists including those studying trafficking and transport in neurons or in other cell types.
All experiments were conducted following approved protocols and according to institutional guidelines for the humane care of research animals. Neonate mice were euthanized by decapitation.
1. Preparation of Microfluidic Devices for Cell Culture
2. Plating of Mouse Hippocampal Primary Neurons in Microfluidic Devices
NOTE: The preparation of cultured hippocampal neurons from neonate rats has been previously published in JoVE25. Below are some modifications that were adapted to plate mouse hippocampal neurons in microfluidic devices, and that were optimal for transfections.
3. Transfection of Hippocampal Neurons Grown in Microfluidic Device
4. “Cargo Mapping” Analyses
Figure 1 shows an overview of the microfluidic device used to grow hippocampal neurons (Figure 1A, B). Neurons are plated in reservoir 1. The size of the microchannels prevents the diffusion of cell bodies (soma) into the axonal compartment while the length of the channels prevents dendritic projections from crossing all the way to the axonal compartment. After ~2-3 days in culture, neurons start extending their axons across microchannels into the axonal compartment (Figure 1B, C). Transfected axons expressing normal prion protein labeled with yellow fluorescent protein (YFP-PrPC) can be identified by fluorescence microscopy (Figure 1C). Expression of fluorescently labeled proteins using plasmid transfection should be checked for proper subcellular localization and function as overexpression might introduce experimental artifacts due to altered protein conformations. Thus, colocalization experiments ought to be complimented both with biochemical co-precipitation studies, as well as with IF staining for endogenously expressed proteins. The behavior of the YFP-PrPC used here was tested previously16. Expression of YFP-PrPC in transiently transfected neuroblastoma cells (N2a) was low, suggesting that transient transfection does not result in highly overexpressed YFP-PrPC levels16. Moreover, IF of N2a cells transfected with YFP-PrPC using antibodies against PrPC indicated that YFP-PrPC highly colocalized with endogenous PrPC, suggesting that both transfected and endogenous PrPC behaved similarly. To ascertain that PrPC associated with motors, vesicle immunoisolations were performed16.
For optimal cargo mapping studies, the right edge of the microchannels is aligned with the field of view during live and fixed imaging in order to image the same region of the axon (indicated by red rectangle in Figure 1B, C). Figure 2 and Figure 3 show representative results for the cargo mapping analyses of neurons transiently transfected with YFP-PrPC. Vesicles carrying YFP-PrPC are transported in mammalian axons by members of the Kinesin-1 family and Dynein Heavy Chain 1 (DYNC1H1)16, therefore YFP-PrPC vesicles were mapped to the Kinesin-1 subunit Kinesin Light Chain 1 (KLC1), and to DYNC1H1 motors. YFP-PrPC vesicle movement was imaged and plotted in a kymograph (Figure 2A). In kymographs, anterograde and retrograde movement of YFP-PrPC vesicles is represented by trajectories with negative and positive slopes, respectively, and stationary vesicles are depicted by vertical trajectories. At fixation, all movement stopped indicated by the absence of diagonal lines in the kymograph (Figure 2A). The IF signal of molecular motors and cargoes in fixed, permeabilized axons is punctate16 (Figure 2B, C). The goal of “cargo mapping” is to determine if puncta in the cargo channel (Figure 2B) colocalize with motor puncta (Figure 2C) in the other two channels. To do this, fluorescent images of fixed YFP-PrPC vesicles, and the IF images of corresponding KLC1 and DYNC1H1 associated to vesicular puncta were aligned to the kymograph (Figure 2B,C). Gaussian functions were fitted to the point spread functions of fluorescent point sources in all three channels in order to map the precise X-Y positions of motors to cargo trajectories obtained by live imaging (Figure 2D). Endogenously expressed KLC1 and DYNC1H1 motors appear as punctate staining and colocalized differentially with YFP-PrPC vesicles (Figure 2D). Colocalization was defined by the presence of YFP-PrPC and motor puncta within a distance of 300 nm and quantified by the level of colocalization of the Gaussian X-Y coordinate positions between the YFP-PrPC and each of the KLC1 and DYNC1H1 puncta (Figure 3A). This distance was chosen based on the optics, diffraction limit of light, and the relevant physical size of the cargo and motor subunits. The relative amount of KLC1 and DYNC1H1 associated with YFP-PrPC vesicles was obtained from the Gaussian amplitudes, which represent the intensities of each puncta (Figure 3B). These data can be further analyzed as desired. For example, intensities of motors colocalizing with YFP-PrPC puncta between control (wild-type – WT) neurons and those lacking other Kinesin-1 subunits (KIF5C -/- neurons in Figure 3A, KIF5C is a member of the Kinesin-1 family), can be compared to determine if the absence of KIF5C disrupts association of KLC1 and/or DYNC1H1 to YFP-PrPC vesicles (KLC1 and DYNC1H1 intensities were unchanged in our experiments16). Furthermore, KLC1 and DYNC1H1 intensities can be plotted to scrutinize possible correlations between relative motor amounts and directionality of movement (Figure 3B). For example, YFP-PrPC vesicles that are moving and/or stationary associated with both KLC1 and/or DYNC1H1 (Figure 3B).
Figure 1. Hippocampal neurons cultured in microfluidic devices. (A) Picture of a microfluidic device. The outlines of reservoirs are visualized by red and blue dyes. Numbering refers to the number of microfluidic reservoirs used throughout the protocol. (B) Diagram of a microfluidic device. Numbers correspond to reservoirs in which media and neurons are added. Transfected neurons are shown in green. Red rectangle and red dotted arrows indicate the region recommended for live and fixed fluorescence imaging. (C) Image of two axons transfected with YFP-PrPC growing through a single microchannel (one microchannel and edge of the device is outlined by dotted white lines). Individually moving YFP-PrPC vesicles can be distinguished as brighter puncta. Scale bar is 10 µm. Figure is adapted from: Encalada et al.16. Please click here to view a larger version of this figure.
Figure 2. “Cargo mapping” to correlate YFP-PrPC vesicle transport with relative amounts of kinesin and dynein motors. Wide-field fluorescence images of live movement and of fixed cargoes were taken on an inverted microscope equipped with a motorized stage and a CCD camera and a 100X/1.4 NA oil objective. (A) Kymograph of YFP-PrPC vesicle movement of axons shown in Figure 1C. Live movement was recorded for a total time ranging from 30 to 60 sec at 100 ms exposure (10 Hz). Cells were fixed after imaging cargo movement for at least 15 sec. Dotted line indicates the time of fixation during live imaging. Red box shows region of vesicles highlighted in (B, C) and enlarged in (D). Arrowheads point to three vesicles shown in (D) corresponding to stationary (blue), retrograde (red), and anterograde (green) trajectories. (B) Image of fixed YFP-PrPC vesicles aligned to the corresponding kymograph. Numbers indicate the individual YFP-PrPC vesicles that were mapped to identifiable puncta on the kymograph. Anterograde vesicles are green, retrograde are red, and stationary ones are blue. (C) Fixed IF images of KLC1 and DYNC1H1 puncta corresponding to the same region shown in (B). Scale bar (A-C) is 10 µm. (D) Enlargement of fixed IF signals of each of the three channels with 2D Gaussian function assignments. Blue dots represent local intensity maxima pixels, red dots represent Gaussian fits, and pink dots are the overlap between the two. Arrowheads indicate examples of YFP-PrPC vesicles shown in (A), and their differential colocalization with KLC1 and DYNC1H1 motor proteins. Scale bar is 2 µm. Figure is adapted from: Encalada et al.16. Please click here to view a larger version of this figure.
Figure 3. Quantitative analyses of “cargo mapping” of YFP-PrPC vesicles. (A) Quantification of colocalization between YFP-PrPC vesicles, KLC1 and DYNC1H1 in WT and KIF5C knockout neurons (KIF5C -/-). Total number of puncta analyzed was NWT= 887 and NKIF5C-/-= 162916. Numbers inside bars represent the number of vesicles per category. Shown are averages ± SEM. Non-parametric permutation t test was used for comparison between genotypes29. (B) Correlation of relative motor composition and directionality of movement in WT neurons of data analyzed in (A). Figure is adapted from: Encalada et al.16. Please click here to view a larger version of this figure.
The protocol presented here enables the correlation of directionality of movement of individual fluorescent microtubule-based moving cargo particles with the relative type and amount of associated motor proteins in live neurons. Previously, the total motor composition of axonal vesicular cargoes was assayed on heterogeneous populations of biochemically purified vesicles and organelles9,15. However, characterizing motor composition for a single type of cargo inside cells has been challenging because of the difficulty in purifying homogeneous vesicle populations. Moreover, while measurements of motor stall-forces provided estimates of active motor numbers in Drosophila embryos, it was unclear whether directionality of movement correlated to motors being stably bound to cargoes or to the rapid association/dissociation of active motors to/from cargo12. Therefore the “cargo mapping” protocol described here was developed to further investigate the correlation between the type and relative number of motors associated to individually moving cargo in live neurons.
To successfully obtain data from “cargo mapping” it is critical to 1) perform imaging of cargo movement and fixed IFs at a high temporal and spatial resolution, 2) ensure immediate fixing of the sample during the course of imaging, 3) align the region of interest in live and fixed images and map fixed cargo and motors to movement kymographs for the same images, and 4) precisely determine the X-Y coordinates of cargo and motor fluorescent puncta in order to determine the amount of colocalization between cargo and motor proteins.
While this protocol is uniquely suited to analyze transport characteristics in uniformly organized axons of neurons grown in microfluidic devices, it could be adapted to a broad range of research questions. For example, this application could be used to characterize association of vesicles with their adaptors involved in endo/exocytosis (e.g., trafficking assays in fibroblasts), or the growth of microtubules labeled with microtubule +tip binding proteins. For these, microfluidic devices could be substituted by gridded coverslips to facilitate the localization of imaged cells necessary for correlative live and fixed imaging studies. This protocol could also be used to characterize cellular events for which a parameter changes in time depending on the specific cellular environment. For example, the release of a fluorescently labeled viral protein from individual endosomal structures into the cytoplasm could be correlated with the expression and/or association of endosomal proteins.
One possible limitation of this protocol is that mapping of high density cargoes might be difficult. However, the assignment of Gaussian functions circumvents this issue to a great extent by allowing distinction of positions between fluorescent puncta at the sub-pixel level. Furthermore, the mapping of motors to axonal cargoes is low-throughput: currently, mapping can be done for 2 axons per microfluidic device, given the low rate of transfection of neurons and the limited field of view when using the high magnification necessary for high-resolution imaging with our camera. Future developments of this method might include the use of lentiviral systems to transduce neurons with higher efficiency, isolation of neurons from transgenic mice expressing fluorescently tagged proteins or the labeling of organelles with small fluorescent dyes such as Lysotracker or Mitotracker, all of which will increase the number of axons labeled with fluorescent markers. Furthermore, the size of the recorded area is limited by the magnification, the pixel size and the size of the pixel array of the camera used for imaging. Ongoing developments in the field of imaging techniques will allow for larger areas to be imaged in the future and therefore increase the amount of data obtained per experimental condition.
The authors have nothing to disclose.
We thank Ge Yang, Gaudenz Danuser, Khuloud Jaqaman, and Daniel Whisler for assistance with the adaptation and development of the software to quantitate cargo mapping analyses, and Emily Niederst for help in making the microfluidic devices. This work was supported in part by NIH-NIA grant AG032180 to L.S.B.G., and the Howard Hughes Medical Institute. L.S. was supported in part by a NIH Bioinformatics Training Grant T32 GM008806, S.E.E. was supported by a Damon Runyon Cancer Research Foundation Fellowship, NIH Neuroplasticity Training Grant AG000216, and by a grant from The Ellison Medical Foundation New Scholar in Aging Award, G.E.C was supported by an NIH/NCATS 1 TL1 award TR001114 and by the Achievement Rewards for College Scientists foundation.
Reagent and Equipment Name | Company | Catalogue Number | Yorumlar |
poly-L-lysine | Sigma | P5899-20mg | |
D-MEM (Dulbecco’s Modified Eagle Medium) High Glucose, w/ L-Glutamine, w/o Sodium Pyruvate (1X) | Life Technologies | 11965092 | |
FBS (Fetal Bovine Serum) | Life Technologies | 10082147 | |
Neurobasal-A Medium (1X) | Life Technologies | 10888022 | |
B-27 Serum Free Supplement | Life Technologies | 10888022 | |
GlutaMAX I Supplement (100X) | Life Technologies | 35050061 | |
HBSS (1X) (Hank’s Balanced Salt Solution) | Life Technologies | 24020117 | |
DPBS (Dulbecco’s Phosphate Buffered Saline, no Magnesium, no Calcium (1X) | Life Technologies | 14190250 | |
Penicillin/Streptomycin (100X) | Life Technologies | 15140122 | |
Corning cellgro Water for Cell Culture | Fisher Scientific | MT46000CM | |
Papain | USB Corporation | 19925 | |
DL-cysteine HCl | Sigma-Aldrich | C9768 | |
BSA (bovine serum albumin) | Sigma-Aldrich | A7906 | |
D-glucose | Sigma-Aldrich | G6152 | |
DNAse I grade II | Roche Applied Sciences | 10104159001 | |
Lipofectamine 2000 | Life Technologies | 11668027 | |
Formaldehyde Solution 16% EM Grade | Fisher Scientific | 50980487 | Caution: Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Dispose according to official regulations. |
Sucrose | Fisher Scientific | S5-500 | |
HEPES | Sigma | H-3375 | |
Normal Donkey Serum | Jackson Immuno Research | 017-000-0121 | |
BSA fatty acid and IgG free | Jackson Immuno Research | 001-000-162 | |
Acetone | Fisher Scientific | BP2403-4 | |
Ethanol | Fisher Scientific | BP2818-100 | |
ProLong Gold antifade reagent | Life Technologies | P36934 | |
Cover Glass 1 1/2. 24X40mm | Corning | 2980-244 | |
Axis microfluidic device, 450 µm | Millipore | AX450 | |
Adobe Photoshop | Adobe | N/A | |
Nikon Eclipse TE2000-U | Nikon | N/A | |
Coolsnap HQ camera | Roper Scientific | N/A | |
60 mm cell culture dish | Fisher Scientific | 12-565-95 | |
150 mm cell culture dish | Fisher Scientific | 12-565-100 | |
Antibodies Used: | |||
Anti-Kinesin light chain, V-17 | Santa Cruz | sc-13362 | specificity verified using KLC1-/- neurons (Ref. 16 ), recommended dilution 1:100. |
Anti Dynein Heavy Chain 1, R-325 | Santa Cruz | sc-9115 | specificity verified using shRNA against DYN1HC1 (Ref. 16), recommended dilution 1:100 |
Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody | Life Technologies | A10042 | recommended dilution 1:200. |
Alexa Fluor 647 Donkey Anti-Rabbit IgG (H+L) Antibody | Life Technologies | A31573 | recommended dilution 1:200. |
Alexa Fluor 568 Donkey Anti-Goat IgG (H+L) Antibody IgG Antibody | Life Technologies | A11057 | recommended dilution 1:200. |
Alexa Fluor 647 Donkey Anti-Goat IgG (H+L) Antibody | Life Technologies | A21447 | recommended dilution 1:200. |
Plugins and Macros | |||
ImageJ | http://imagej.nih.gov/ij/index.html. | ||
ImageJ Kymoraph Plugin | http://www.embl.de/eamnet/html/body_kymograph.html. |