Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans

Summary

The paper describes the optimization of fluorescence microscopy acquisition parameters to visualize the axonal transport of endogenous labeled cargos at single-neuron resolution in a living nematode.

Abstract

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).

Introduction

Throughout life, neurons rely on axonal transport to deliver proteins, lipids, and other molecules from the cell body to their final destination in the axon. Consequently, impairment of axonal transport is associated with a loss of neuronal function and is often involved in the pathology of neurodegenerative disorders^1,2. Hence, understanding the mechanisms that underly axonal transport is of great interest.

Several decades of research on axonal transport revealed many important insights into the molecular machinery that mediates this transport, their composition as well as regulatory mechanisms. Long-range axonal transport occurs on the microtubule cytoskeleton, which consists of partially overlapping microtubule polymers that are typically oriented with their plus end out in axons³. Consequently, anterograde transport is mediated by motor proteins that walk to the plus end of microtubules, kinesins, whereas retrograde transport depends on the minus end directed dynein motor. Although many aspects of transport have been revealed, for many axonal proteins it still remains unclear, how they are loaded into the transport machinery, how individual transport packages are organized, and how this transport is regulated³.

Axonal transport was initially studied in radio-labeling experiments, in which radiolabeled amino acids were injected into the somatic compartment, where they were incorporated into nascent endogenous proteins and could be traced over time in the axonal compartment by autoradiography⁴. Although radiolabeling experiments allowed the study of axonal transport of endogenous proteins in vivo, it does not allow for the direct follow-up of the behavior of individual cargo to get mechanistic insights⁴. This limitation was overcome with the use of fluorescence microscopy. However, axonal transport is often not visualized on endogenous proteins but instead by expression of a fluorescent labeled copy. Especially for low expressed proteins, overexpression provides higher signal intensities which make visualization, preferably with single neuron resolution, possible. Moreover, ectopic expression of the fluorescent tagged protein circumvents the need and challenges of genome editing. Conversely, it has been argued that the behavior of ectopically expressed cargo may differ from the behavior of the endogenous cargo⁵.

Recent improvements in genome editing made endogenous labeling strategies easier accessible. Hence, a lower signal intensity has become the major limitation to study axonal transport of a cargo by ectopic expression instead of endogenous labeling. Careful considerations in the endogenous labeling strategy paired with an optimization of the acquisition conditions can overcome this challenge.

Nematodes provide an excellent research model to study axonal transport in vivo due to their transparency and ease in genetic manipulations. In this protocol, we describe a research strategy to visualize axonal transport of endogenous proteins with single neuron resolution in living Caenorhabditis elegans. We visualize the axonal transport of synaptic vesicle precursors by using a strain generated by the Jorgensen Lab⁶, in which the vesicle associated RAB GTPase, RAB3, is endogenously labeled with GFP, in the motor neuron DA9. By asking how small adaptations in different acquisition parameters and photobleaching can improve the visualization of individual transport events, the protocol provides ideas on how to optimize imaging conditions.

Protocol

For a detailed protocol on how to maintain and prepare nematodes for live-cell imaging, refer to the work of S.Niwa ⁷.

1. Worm strain generation

In addition to generating nematode strains, the Caenorhabditis Genetics Center (CGC)⁸ contains a growing collection of nematode strains with endogenously fluorescently tagged proteins that can be directly obtained from their webpage.

1. Choice of the fluorescence labeling strategy
   1. Use the nematode strain MTS1161, which contains the rab-3(ox699) allele⁶, to visualize endogenous GFP labeled RAB-3 in the DA9 motor neuron (Strain will be deposited at the CGC). To visualize other axonal proteins, generate a nematode strain with an endogenous tagged protein by using the CRISPR Cas9 editing protocol from the Mello Lab⁹.
      NOTE: MTS1161 utilizes a recombination approach to cell specifically label RAB-3 endogenously with a GFP tag⁶. A common alternative approach is based on the reconstitution of a split-fluorescent protein, which is recommended for especially low expressed proteins as it enhances the fluorescent copy number per endogenous protein by using multiple split fluorescent copies¹⁰. The copy number can initially be estimated based on transcriptome datasets from the CenGen webpage (https://cengen.shinyapps.io/CengenApp/) ​¹¹.
2. Choice of neuron to visualize axonal transport
   1. In the strain MTS1161, visualize RAB-3 in the motor neuron DA9 (see Figure 1). For other axonal cargos, especially low expressed proteins, identify a neuron in which the expression levels of the analyzed protein are highest using the CenGen webpage.
      NOTE: The CenGen project (cengen.org) provides a great online resource to estimate expression levels for a protein of interest in many neurons of the nematode based on a large transcriptomic dataset covering all 302 neurons of the C. elegans nervous system^12,13.

2. Worm handling and preparation for imaging

1. Maintain nematodes on a lawn of bacteria (strain: OP50) seeded on nematode growth medium plates at 20 °C and grown to an age of 1 day into adulthood for imaging.
   NOTE: Measuring axonal transport at a defined age stage is important as transport rates declines with ageing consistently across different axonal cargos, neuron classes and organisms^{14,15,16,17,18}-Nematodes at larval stage L4 or 1 day into adulthood have been used in most papers and thus offer the largest datasets for comparisons.
2. Prepare nematodes for live cell imaging: Perform all subsequent steps with a stereo microscope to visualize and handle the worms.
   1. Transfer nematodes to a droplet of 0.5 mM Levamisole using a platinum wire pick and incubate the nematodes for 20 min in the droplet.
   2. Carefully mount 10-20 nematodes from the Levamisole droplet into a 12 µL droplet of M9 medium on a 10% agarose (dissolved in M9 medium) patch on a glass slide by using a hair pick. For a detailed procedure on how to make a 10% agarose patch, refer to the protocol of S.Niwa⁷.
      NOTE: Mounting a low number of nematodes is sufficient since only a few animals will be imaged per slide before the animals start to suffer from hypoxia.
   3. Place a cover slip on top of the droplet (22 mm x 22 mm or 18 mm x 18 mm). For imaging transport in the DA9 axon, position the axon close to the cover slip. To do so, rotate the cover slip by 45° after placing it on top of the agar patch, to roll the animals onto their back.
   4. Seal the space between the cover slip and the glass slide with a viscous gel like petrolatum. This will prevent the agarose patch from drying, which can cause the nematodes to drift out of the imaging field.
      NOTE: It is crucial to treat the animals as gently as possible. Too high concentrations of levamisole or physical damage to the worm can impair axonal transport. Mild twitches of the tail during the imaging session are a good sign that the animal remains in a healthy state. Animals remain healthy and can even recover from the agarose patch after the imaging session.

3. Live-cell Microscopy

NOTE: Exact acquisition parameter values can differ between microscopes. However, trends for each acquisition parameter should be independent of the microscope used. A spinning-disc confocal microscope that was equipped with a separate laser line for bleaching was used in this protocol (see Table of Materials for details on the microscope). Green fluorescence was excited by a 488 nm laser and emission was filtered by an ET525/36 emission filter. Bleaching was performed using a 488 nm laser line.

1. Ensure that the temperature of the room, or if a temperature-controlled stage is available, is set to a constant temperature. For nematodes, set the temperature to 20 °C.
2. Mount the slide and use objective lens with 4x-20x magnification to locate the nematodes on the slide and memorize positions. Switch to 63x magnification lens for image acquisition.
3. Take a single image to check initial acquisition parameters. Track the signal intensities in the field of view with the intensity histogram of the acquisition software. Intensity values should cover the lowest third of the maximum signal that the camera can detect. Avoid pixel saturation. Enhance the intensity of the excitation laser if the signal intensity is too low.
   NOTE: Do not use too high excitation laser intensities as it bleaches the fluorescent protein during time-lapse.
4. Alter the acquisition parameter and steps here to optimize fluorescence signal detection.
   1. Implementation of a bleaching step: Bleach the region of the axon that is going to be analyzed using a 488 nm laser (power:15 mW) line for the bleaching step. Aim for a bleaching efficiency of at least 90%. Determine the bleaching efficiency by comparing the signal intensity of the region before and after the bleaching step.
      NOTE: Bleaching enhances the signal of moving particles by lowering the signal that derives from stationary particles as well as from the signal that derives from the cytoplasmic fraction of the protein in the background. Membranous cargo, such as RAB-3 on synaptic vesicle precursors have a low cytoplasmic fraction so that the bleaching step only mildly improves the signal of the transport package over the cytoplasmic background (Figure 2A,D). However, bleaching improves tracing individual transport events over longer distances and allows quantifying pausing times (Figure 2A).
   2. Binning: Use 2 x 2 binning to enhance the signal intensity of individual transport events. Binning combines pixel arrays into one larger pixel. This will reduce the spatial resolution but enhance the signal intensity of individual transport events and is especially helpful for dim particles (Figure 2B,D).
   3. Exposure time: Enhance the exposure time to enhance the signal intensity. Enhancing the exposure time enhances the signal intensity of the sample to the cost of getting a lower temporal resolution for the transport events. For the experiment here, use a temporal resolution of 300 ms to 700 ms between consecutive timepoints (100- 500 ms exposure time) to follow individual transport events of RAB-3. (Figure 2C,D)
5. Time-lapse recording: Take a time lapse of at least 1-3 min depending on the signal intensity of the protein as well as the frequency of individual transport events. Once a time lapse has been recorded move to the next animal. Animals on a slide should not be imaged for more than 30 min to ensure that the recorded animals are in a healthy state.
   NOTE: Mild twitches of the animals' tail during the recording are an indicator that the animal is still alive.

4. Analyzing axonal transport data

NOTE: Use ImageJ/Fiji¹⁹ for the subsequent image analysis steps. Fiji is able to read data by all common microscopy software packages.

1. Kymograph generation
   1. Import data: Import the time-lapse data file into Fiji. In case it cannot import the data into Fiji, export the time-recording as a tiff file from the software package and load it subsequently into Fiji.
   2. Drift correction: Check whether the animal/axon segment moved or slightly drifted during the image acquisition. Correct for animal movement or drifting by running the plugin StackReg²⁰. When running the plugin, choose the Rigid Body transformation.
   3. Manual generation of a kymograph: A detailed step by step procedure is provided in Figure 3. Utilize the drift corrected movie to generate the kymograph. Use the Segmented Line tool and adjust the line width to match the axon diameter by double clicking with the left mouse button on the segmented line icon to draw a line along the axon segment that will be analyzed. Run the ImageJ/Fiji plugin Kymoreslicewide to generate the kymograph and utilize the maximum intensity value across the width of the line in its parameter settings.
      NOTE: Keeping consistency in the line drawing direction (e.g., always proximal to distal axon or reverse) simplifies tracing back movement direction in the kymographs.
2. Analysis of transport parameter
   1. Calculate the transport parameter: event number, velocity, run length and pause duration from the kymograph by following the subsequent steps.
      1. Trace transport events in the kymograph: Use the Straight Line tool to trace individual transport events on the kymograph. Save each line to the ROI manager and trace all transport events in the kymograph. Choose the following measurement parameters in ImageJ/Fiji: Area, Bounding rectangle, Mean gray value, Feret´s diameter.
      2. Calculate transport parameter: Paste the results table into a spreadsheet and use the following columns of the result sections to calculate:
         Run length: Multiply width (in pixel) by the resolution of the camera (e.g., x µm/pxl) to determine the run length in µm.
         Velocity: Run length/Run duration. To determine the duration of a movement event in seconds, multiply height (in pixel) by the acquisition time between consecutive timepoints (e.g., x s/pixel).
         Pausing time: Run duration in between two consecutive movements at which velocity is 0.
         Event number: Total events can be normalized to the total length of the kymograph to determine event number per min and axon length segment. Events can further be categorized into anterograde and retrograde transport events. To determine the directionality of each movement event, utilize the Feret Angle tool. In kymographs that were initially generated by drawing the segmented line from proximal to distal and the anterograde transport events in the kymograph are pointing from right to left, a Feret Angle < 90° will indicate an anterograde and >90° a retrograde event, otherwise it will be the inverse.

Representative Results

Overview of the model system and measurement procedure
To visualize axonal transport of synaptic vesicle precursors, we traced endogenously GFP labeled RAB-3. Here we make use of a recently generated GFP::Flip-on::RAB-3 strain⁶, in which expression of the recombinase Flippase under a cell specific promoter (glr-4p) labels endogenous RAB-3 in the DA9 motor neuron. DA9 is a bipolar motor neuron, with its cell body located in the posterior of the animal on the ventral side, close to the anus (Figure 1A). It contains a short dendrite that runs anterior along the ventral nerve cord and a long axon that runs posterior, forms a commissure and then runs anterior along the dorsal nerve cord, where it forms en passant synapses that innervate the dorsal muscle and VD neuron^22,23,24. We visualize axonal transport in the asynaptic region; most transport events can be captured with a single focal plane (Figure 1A). An initial photo-bleaching step is implemented to reduce the background of stationary vesicles in this area, which often tend to pause in this region (Figure 1B). Time-lapse videos are recorded over 3 min and the RAB-3 signal along the asynaptic region is plotted into a kymograph to extract individual transport events (Figure 1C).

Adjusting acquisition parameter to optimize axonal transport visualization
To overcome the low signal intensity of many endogenously fluorescent tagged proteins, acquisition parameters of the microscope need to be optimized. For a robust quantification of transport events, the signal intensity of individual transport events needs to be as bright as possible over the cytoplasmic background or pausing, stationary cargo. Synaptic vesicle precursors often pause in the asynaptic region in distinct vesicle pools, which interfere with the detection of novel incoming vesicles and makes it difficult to follow their transport traces⁷.

A single initial fluorescence bleaching step can strongly reduce the fluorescence signal deriving from the vesicle pools to enhance the movement detection of novel incoming vesicles (Figure 2A). The bleaching step only mildly enhanced the signal of moving RAB-3 vesicles over the cytoplasmic background intensity likely because the cytoplasmic fraction of RAB-3 is very low (Figure 2D).

The use of binning provides an additional layer to improve the signal over background of individual transport events by combining an array of pixels into a single pixel. A 2 x 2 binning will halve the spatial resolution (here from 108.33 nm/pixel to 216.7 nm/pixel), which is still sufficient to trace individual RAB-3 transport particles (Figure 2B) but strongly improves the signal intensity of individual vesicles (Figure 2D). Unless very high spatial resolution is required, binning can also be applied to visualize many other axonal cargos.

Next, we asked how changes in the exposure time can improve the signal intensity of single transport events. We especially included exposure time in the analysis to also demonstrate that up to 500 ms exposure time with 700 ms between subsequent imaging time points still provides a temporal resolution at which synaptic vesicle precursors can be tracked (Figure 2C,D).

Generating and analyzing kymographs with Fiji
To generate a kymograph and analyze individual transport events following steps in Figure 3.

Figure 1: Overview of the model system to visualize axonal transport of endogenous fluorescent RAB-3. (A) Upper panel: Overview of the nematode with indicated posterior-anterior and ventral-dorsal axis. The motor neuron DA9 is labeled blue. Mid panel: Zoom in of the boxed region shown in the upper panel with indicated DA9 compartmentalization. En passant synapses are illustrated in green, * indicates the anus. Note that the axon continues in the dorsal nerve cord until it passes the vulva. The red box indicates the region of the asynaptic zone. Lower panel: Confocal microscopy image of endogenous GFP labeled RAB-3 in the proximal axon in a single focal plane. Note that there are vesicle sinks of longer pausing RAB-3 in the asynaptic region, although most RAB-3 signal clusters in the synaptic region. Asynaptic region is boxed in red. Scale: 20 µm. (B) Representative images of a time-lapse recording to visualize axonal transport. To enhance the traceability of individual transport particles over stationary particles, the asynaptic region is initially photobleached. Lower panels in purple boxed region show an example of an anterograde (orange arrowhead) and retrograde (blue arrowhead) movement event. Panels from top to bottom represent consecutive timepoints. (C) Time-lapse recording in (B) was plotted as a kymograph (2D representation of time over position). Diagonal lines represent movement events in which the slope indicates the velocity. Vertical lines show stationary events. Purple box is a zoom in of the kymograph to highlight the transport events that are also shown in (B) and the anterograde (orange) and retrograde (blue) transport events are traced. Dashed vertical lines indicate the pausing events. Please click here to view a larger version of this figure.

Figure 2: Optimizing acquisition steps and parameters to improve the visualization of endogenous axonal cargo. Endogenous GFP::RAB-3 were visualized in the asynaptic zone of the DA9 axon taking images of a single focal plane on a confocal spinning disc microscope. Kymographs were acquired to visualize individual transport events in anterograde (right to left) and retrograde (left to right) direction (A-C). (A) Kymographs show RAB3 transport events without (left kymographs) or with an integrated bleaching step. Note that the bleaching step improves the visualization of pausing events (indicated by yellow arrow heads) between consecutive transport events (orange arrowheads). (B) Implementing 2 x 2 binning helps to improve the signal intensity of faint RAB3 transport events. Images were acquired at 300 ms exposure time and with (C) incremental increase of the exposure time (from 100 ms on the far left to 500 ms on the far right) helps to improve the signal intensity of individual transport events. Images were acquired at 2 x 2 binning and using an initial photobleaching step. All kymographs display a total duration of 3 min and are drawn at the same scale so that kymographs with less acquisition time points due to a longer time lapse between consecutive imaging points have a shorter total length of the kymograph. (D) Quantification of the signal intensity per transport event after subtraction of background fluorescence from kymographs in (A-C). Note that binning and the photo bleach step were acquired at 300 ms exposure time. Statistical comparison using n events for 100 ms (n_events= 27 in n_animals= 2), 200 ms (n_events= 30 in n_animals= 2), 300 ms (n_events= 88 in n_animals= 6), 500 ms (n_events= 12 in n_animals= 1), 300 ms without (w.o.) binning (n_events= 31 in n_animals= 3), 300 ms with (w) binning (n_events= 88 in n_animals= 6) and 300 ms, 2 x 2 binning with bleaching (n_events= 88 in n_animals= 6) and without bleaching. Each datapoint represents the signal intensity of an individual RAB-3 transport event after background (cytoplasm of the axon) subtraction at a single timepoint along its trace that was chosen randomly. Exposure time was compared using a Kruskal-Wallis test followed by Dunn´s multiple comparison, binning and photo bleaching were compared using a pairwise Mann-Whitney test. Error bars indicate standard deviation. Please click here to view a larger version of this figure.

Figure 3: Step by step procedure to generate a kymograph and measure individual transport events. (A) After loading the video recording into Fiji, the segmented line tool is used to trace a line along the length of the axonal segment that is going to be analyzed. (B) A kymograph (right panel) is generated using the Kymoreslicewide plugin with the parameters depicted in the left panel. (C) Individual transport events are traced with the linear line tool and stored to the ROI manager. The right panel show the measurement settings that are used to generate the results table. (D) Results in the table are used to calculate transport parameter. Boxes indicate important parameters that are described in the methods section of the protocol. Please click here to view a larger version of this figure.

Discussion

Limitations of the method and alternative methods
In this protocol, we optimized acquisition parameters to visualize the axonal transport of endogenously tagged RAB-3, which is associated with synaptic vesicle precursors. To visualize RAB-3, we made use of a recently published FLIP-on::GFP::RAB-3 strain⁶ and expressed the recombinase Flippase under a cell specific promoter (glr-4p)²⁵. This strategy allows us to label RAB-3 with a single GFP fluorophore. RAB-3 is relatively easy to visualize because it is highly expressed and strongly enriched on synaptic vesicle precursors so that individual transport events have a bright signal intensity over a low signal that derives from the cytoplasmic background. Other axonal proteins might require additional optimization strategies outside of the microscopy settings to enable the visualization of transport events. Especially for low expressed proteins, the split-GFP system provides a great alternative. In this system, GFP₁₁ is inserted into the endogenous locus of the protein of interest and GFP_1-10 is expressed from a cell specific promoter. A great advantage of this system is the small size of the DNA repair template (approximately 70 nucleotides) that needs to be genomically inserted, which makes homologous recombination more efficient compared to inserting the ORF of the entire fluorescent tag^26,27. Because of its small size, multiple GFP₁₁ fragments can be inserted to the endogenous protein, which amplifies the number of reconstituted GFP fluorophores per protein and thus enhances the fluorescent signal of the endogenous protein and thus of the transport package²⁸.

In addition to labeling the protein with a split-GFP or GFP approach, other genetically encoded fluorophores can be utilized that have unique advantages and disadvantages regarding their photochemical properties, which have been recently compared²⁹. Moreover, chemical fluorophores with more photostable properties can be used by endogenously labeling the protein of interest with a HALO or SNAP tag³⁰.

Especially for cytoplasmic proteins that are abundant throughout the entire axon, a conditional labeling approach might be necessary. We recently visualized such a cargo, endogenous spectrin, by fluorescence microscopy using temporally controlled labeling to only visualize novel synthesized spectrin proteins. For conditional labeling with single neuron resolution, we combined heat-shock driven expression of a recombinase with a split-GFP system³¹.

If the research goal aims to understand the transport of organelles, expression of a protein domain that associates with the organelle can provide an alternative to ectopically expressing the full-length protein.

Critical steps in the protocol
A careful preparation to optimize the visualization of the protein based on expected expression levels as well as to choose the optimal labeling strategy are especially critical for the successful visualization of endogenous labeled proteins. Expression levels for most protein in many neurons can be estimated based on the transcriptomic dataset from Cengen¹³. Expected low signal intensity of transport events for low expressed or cytoplasmic proteins can be improved by attaching multiple copies of the fluorophore. Note, however, that with any labeling experiment, care must be taken not to disrupt the function of the tagged protein. In case there is a known phenotype for the corresponding mutant, it is important to verify that labeling does not generate this phenotype. In cases where there is no known phenotype, alternative approaches are required which would vary on a case-by-case basis.

A critical step in the imaging of axonal transport events is the health state of the animal. The animals should be treated as gently as possible for image preparation as well as during imaging (e.g., use of a hairpick instead of a metal wire to transfer paralyzed animals, minimizing incubation time in the paralyzing agent, minimize imaging time to avoid phototoxicity).

Modifications and troubleshooting
While we intend to optimize axonal transport visualization of endogenous proteins, the optimization strategies for acquisition parameters are also applicable to ectopic expressed proteins. Especially if the endogenous expression levels are too low to visualize transport events, ectopic expression of the protein can provide an alternative.

In case no transport events can be recorded despite a strong signal intensity of the endogenous labeled protein, the animals might be in an unhealthy state. This can be solved by preparing animals for microscopy under more gentle preparation procedures (e.g., lower the incubation time in the paralyzing agent). Alternatively, transport events might be rare, and a 3 min video recording might not be sufficient to capture events. Capturing rare transport events can be optimized by enhancing the duration of the video recording, by enhancing the number of imaged animals or by imaging the animals at a different developmental stage at which transport events might be more frequent.

Materials

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A9539
Cover slips (22 mm x 22 mm, No1); Gold Seal Cover Glass	Thomas Scientific	6672A14
Levamisole	ChemCruz	sc-205730
Microscope: Nikon Ti2 inverted microscope, Yokogawa CSU-W1 SoRa Scanhead, Hamatsu Orca-Fusion BT sCMOS camera, Nikon CFI Plan Apo lambda 60x 1.4 NA oil immersion objective, Nikon photostimulation scanner at 488nm with an ET525/36 emission filter	Nikon		Spinning Disc Confocal Microscope
NIS-elements AR	Nikon		Software for the Nikon Ti2
Plain precleaned microscopy slides	Thermo Scientific	420-004T
Nematode strain	Identifier	Source
rab-3(ox699[GFP::flip-on::rab-3]) (II); shyIs43(glr-4p::FLP-NLSx2; odr-1p::RFP) (II)	Park et al. (DOI: 10.1016/j.cub.2023.07.052)	MTS1161	Will be deposited at CGC (https://cgc.umn.edu/)