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. 2021:2259:25-45.
doi: 10.1007/978-1-0716-1178-4_3.

Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions

Ana Gordon  1   2 , Karine Gousset  3
Affiliations

Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions

Ana Gordon et al. Methods Mol Biol. 2021.

Abstract

Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small amounts of samples. Although cellular protrusions-such as tunneling nanotubes, filopodia, growth cones, invadopodia, etc.-are involved in essential physiological and pathological actions such as phagocytosis or cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and transient nature. The method described herein, combining LCM and MS, has been designed to identify the proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient protein recovery and high sample quality of this method enable the protein profiling of these small and diverse subcellular structures.

Keywords: Cellular protrusion; DTBP; Fixation; Laser capture microdissection; Mass spectrometry; Proteomics.

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Figures

Fig. 1.
Fig. 1.. Effect of fixations, protein extractions and dehydration of samples on protein yield.
GLU/PFA fixed samples (A) lysed with RIPA buffer (0.01% SDS) on ice or (B) lysed with RIPA buffer (2% SDS) and incubations at 100°C/20 min and 60°C/2h; (1) Unfixed lysates, (2) 4% PFA alone, (3) GLU (0.05%)/PFA, (4) GLU (0.01%)/PFA, (5) GLU (0.005%)/PFA; (C) PFA/DTBP fixed samples were lysed with RIPA buffer (2% SDS and 100 mM DTT) and incubations at 37°C/30 min, 100°C/20 min and 60°C/2h; (1) Unfixed lysates, (2) 4% PFA alone, (3) PFA/3mM DTBP, (4) PFA/5mM DTBP, (5) PFA/10mM DTBP. (D) PFA/5mM DTBP fixed samples extracted and loaded as in gel C; (1) Cells were fixed and lysed the same day; (2) Cells were fixed and kept in PBS for 24h before lysing; (3) Cells were fixed and kept dry for 24h before lysing; (4) Cells were fixed and kept in PBS for 72h before lysing; (5) Cells were fixed and kept dry for 72h before lysing. Stars indicate protein bands with decreased intensity. Gels are representative of 3 independent experiments (reproduced from ref. with permission from Proteomics).
Fig. 2.
Fig. 2.. Effect of type of fixation on protein yield and MS results from 1,000 cells.
(A) Silver-stained representative gel from (1) Unfixed cells (0.1 μg of protein); (2) 1,000 GLU/PFA fixed cells; (3) 1,000 PFA/DTBP fixed cells; (B) Average and Standard Deviation of unique proteins, unique spectra and unique peptides of samples from 1,000 PFA/DTBP or GLU/PFA fixed cells (reproduced from ref. with permission from Proteomics).
Fig. 3.
Fig. 3.. Cellular protrusions isolated by Laser Capture Microdissection.
LCM, which uses a finely focused laser to cut a region of interest (ROI), is ideal to isolate cellular protrusions. Cells were plated on MMI live cell chambers, fixed, and imaged at ×20 (A), ×40 (B–D) or ×60 (E) magnifications. Various types of cellular protrusions are shown. For all cases, (i) ROIs are drawn around the protrusions of interest; (ii) are representative images of cellular protrusions after the laser cut, and (iii) images of the desired isolated cellular protrusions after removal of the LCM membrane containing the “unwanted” cells. Examples of different types of protrusions are shown: (A) axons and dendrites from dCADs are shown (scale bar = 100 μm). Small dendritic filopodia can be observed (black arrows); (B) in order to increase the number of cellular protrusions, CADs cells were treated for 5 min with 100 μM H2O2 prior to fixation. Various types of cellular protrusions are shown (scale bar = 10 μm). Individual subtypes of cellular protrusions can be specifically isolated such as (C) GCs (scale bar = 20 μm), (D) filopodia (scale bar = 10 μm), and (E) TNTs (scale bar top = 50 μm; bottom = 10 μm). TNTs do not touch the substratum and tension is visible within these structures (Ei). As expected, after being cut, the structures collapsed onto the LCM membrane (Eii,iii), clearly demonstrating that these protrusions were TNTs, and not attached filopodia or other types of protrusions (reproduced from ref. with permission from International Journal of Molecular Science).
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