Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Summary

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

Abstract

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley’s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Introduction

Exocytosis is a universal cellular process in which a vesicle fuses with the plasma membrane and releases its content. The vesicle can either fuse totally with the plasma membrane (full fusion) or create a fusion pore that stays open during a limited time (kiss-and-run)¹. For instance, newly synthesized proteins are released into the extracellular medium from vesicles that come from the Golgi complex. This biosynthetic, anterograde pathway is primordial, especially in multicellular organisms, to secrete signaling peptides (e.g., hormones, neurotransmitters) and extracellular matrix components (e.g., collagen), as well as to traffic transmembran....

Protocol

1. Preparation of micropatterned cells

1. Transfection of cells
   1. One day before transfection, seed 2.5 x 10⁶ hTERT-RPE1 cells into one well of a 12 well plate (2 x 2 cm) in 1 mL of medium.
   2. On the day of transfection, prepare the transfection mixture with VAMP7-pHluorin plasmid (100 µL of buffer, 0.8 µg of DNA, 3 µL of transfection mixture). Incubate for 10 min.
      NOTE: VAMP7 is a lysosomal v-SNARE, fused with a luminal pHluorin tag. The pHluorin probe is quenched by low pH, but during exocytosis protons are released and pHluorin starts emitting a signal^10,

Results

The spatiotemporal characteristics of exocytosis events were analyzed from lysosomes visualized by VAMP7-pHluorin^10,11 in hTert-RPE1 cells. hTert-RPE1 cells are nontransformed cells that adopt well to micropatterning and have been extensively used in previous micropattern-based studies^4,14. VAMP7 is a lysosomal v-SNARE¹⁵ that was tagged with the super ecliptic pHluorin at its N-ter.......

Discussion

We monitored exocytosis events from the lysosomal compartment by TIRFM live cell imaging of VAMP7-pHluorin in ring-shaped micropattern-normalized cells and performed a rigorous statistical analysis of the spatial parameters of exocytosis events. Employing the transformed Ripley’s K function and a statistical test based on the nearest neighbor distance, we confirmed that secretion from lysosomes is not a random process^8,9. Both statistical analyses convincin.......

Materials

Name	Company	Catalog Number	Comments
Chamlide Magnetic Chamber	Chamlide
DMEM/F12	Gibco	21041-025
Fibrinogen	Molecular Probes, Invitrogen	F35200
Fibronectin bovine plasma	Sigma	F1141
HEPES (1M)	Gibco	15630-056
hTert RPE1 cell line	https://www.atcc.org
ImageJ	http://rsbweb.nih.gov/ij/	n/a	Authored by W. Rasband, NIH/NIMH
JetPRIME Transfection reagent	Polyplus	114-07
Penicilin/Streptomycin	Gibco	15140-122
Photomask	Delta Mask
PLL-g-PEG solution	Surface Solutions	PLL(20)-g[3.5]- PEG(2)
R Software	https://www.r-project.org/	n/a
Trypsin (TrypLE Express 1X)	Gibco	12605-010
UV ozone oven	Jelight Company Inc	342-220
VAMP7-pHFluorin plasmid	n/a	n/a	Paper reference :http://www.ncbi.nlm.nih.gov/pubmed/?term=Role+of+HRB+in+clathrin-dependent+endocytosis. J Biol Chem. 2008 Dec 5;283(49):34365-73. doi: 10.1074/jbc.M804587200. Role of HRB in clathrin-dependent endocytosis. Chaineau M, Danglot L, Proux-Gillardeaux V, Galli T.