3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Summary

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Abstract

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology.  To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest. 

Introduction

Cells organize to form functional structures through the coordination of intra- and intercellular changes via morphogenesis. A remarkable example of morphogenesis is the development of the highly specialized neuronal structure. Neurons display remarkable polarization, in which they extend two structurally and functionally distinct types of processes, dendrites and axons¹, which can achieve immense lengths. The complexity of neuronal development arises not only from the sheer size of dendrites and axons but also from the difficulty in forming their intricately branched geometries^2,3. Neu....

Protocol

1. Generation of Drosophila specimens

1. Select a suitable MT plus-end marker. This study utilized GFP-tagged EB1, a well-characterized plus-end marker with a strong, clear signal^11,12. Alternatives include other +TIPs such as EB3^10,13, CLASP/Orbit⁵³, and CLIP-170⁵⁴.
2. Obtain or generate flies with the MT marker under control of a UAS promot.......

Discussion

This paper discusses a protocol to perform noninvasive intravital imaging of MT dynamics in the dendrites and at the NMJ of during development. Human input is required during the experimental steps, such as in selecting animals to image, and may introduce bias in the data collection process that cannot be reasonably removed. Thus, a key goal of the protocol is to minimize bias wherever possible by performing automated analysis with a new software (section 5) that was optimized to handle the low signal-to-noise ratio inhe.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	Eppendorf	21008-959	Sample preparation
1000 µL TipOne pipette tips	USA Scientific	1111-2721	Sample preparation
200 µL TipOne pipette tips	USA Scientific	1120-8710	Sample preparation
221-Gal4 flies	Bloomington Drosophila Stock Center (US)	26259	Drosophila genetics/crosses
60x Objective Lens	Nikon	Plan Apo 60x Oil	Image acquisition
6-well plate	BD Falcon	353224	Sample preparation
Agar	MoorAgar	41084	Drosophila food
Aivia	DRVision LLC		Optimized as part of this study
Chloroform (stabilized with amylenes)	Sigma-Aldrich	C2432	Sample preparation
CO2 blowgun (for selection of flies for crosses)	Genesee	54-104	Drosophila genetics/crosses
CO2 bubbler (for selection of flies for crosses)	Genesee	59-180	Drosophila genetics/crosses
Cooled CCD camera	Hamamatsu	ORCA-R2	Image acquisition
Cornmeal	Genesee	62-101	Drosophila food
Distilled Water			Drosophila food
Double-sided tape	Scotch		Sample preparation
Drosophila vials	Genesee	32-109	Drosophila food
Droso-plugs (foam plugs for vials)	Genesee	59-200	Drosophila food
Dumont #5 Biologie Inox Forceps	Fine Science Tools	11252-20	Sample preparation
elaV-Gal4;UAS-EB1-GFP;UAS-Dcr2 flies	Gift of Melissa Rolls (Penn State University)	N/A	Drosophila genetics/crosses
Ethanol (95%)	VWR	75811-022	Drosophila food
Fiber optic illuminator/light source for stereomicroscope	Nikon	NI-150	Sample preparation
Flypad (for selection of flies for crosses)	Genesee	59-172	Drosophila genetics/crosses
Forma Environmental Chamber/Incubator	ThermoFisher	3940	Drosophila genetics/crosses
Halocarbon oil 700	Sigma-Aldrich	H8898	Sample preparation
Immersion Oil	Nikon	MXA22168	Image acquisition
Kimwipe Delicate Wipes	Fisher Scientific	34120	Sample preparation
Laser Merge Module	Spectral Applied Research	LMM-5	Image acquisition
Light Source for Confocal	Lumencor	SOLA 54-10021	Image acquisition
MetaMorph Microscopy Automation & Image Analysis Software	Molecular Devices		Image acquisition
Micro Cover Glasses, Square, No. 1 1/2 (#1.5)	VWR	48366-205	Sample preparation
Motorized inverted microscope with Perfect Focus System	Nikon	TI-ND6-PFS-S	Image acquisition
Motorized stage and shutters	Prior	Proscan III	Image acquisition
Multi-purpose scissors	Scotch	MMM1428	Sample preparation
Nail Polish	Sally Hansen	784179032016 074170382839	Sample preparation
Optical Filter	Chroma	ET480/40m	Image acquisition
P1000 Pipetman	Gilson	F123602	Sample preparation
P200 Pipetman	Gilson	F123601	Sample preparation
PBS (10X) ph 7.4	ThermoFisher	70011044	Sample preparation
Propionic Acid	Fisher	A258-500	Drosophila food
Spinning disk confocal scanner unit	Yokagawa	CSU-X1	Image acquisition
Stereomicroscope	Nikon	SMZ800N	Sample preparation
Sugar (Sucrose)	Genesee	62-112	Drosophila food
Superfrost Slide	VWR	48311-600	Sample preparation
Tegosept	Genesee	20-258	Drosophila food
UAS-dtacc-RNAi flies	Vienna Drosophila Resource Center (Vienna, Austria)	VDRC-101439	Drosophila genetics/crosses
Vaseline petroleum jelly	WB Mason	DVOCB311003	Sample preparation
Winsor & Newton Brush Regency Gold 520, Size 0	Staples	5012000	Drosophila genetics/crosses
Yeast	VWR	Torula Yeast IC90308580	Drosophila food
Yokogawa dichroic beamsplitter	Semrock	Di01-T405/488/568/647-13x15x0.5	Image acquisition