Spatio-Temporal Analysis of Constitutive Exocytosis in Epithelial Cells

Abstract

Exocytosis is an essential cellular trafficking process integral to the proper distribution and function of a plethora of molecules, including transporters, receptors, and enzymes. Moreover, incorrect protein targeting can lead to pathological conditions. Recently, the application of evanescent wave microscopy has allowed us to image the final steps of exocytosis. However, spatio-temporal analysis of fusion of constitutive vesicular traffic with the plasma membrane has not been systematically performed. Also, the spatial sites and times of vesicle fusion have not yet been analyzed together. In addition, more formal tests are required in testing biological hypotheses, rather than visual inspection combined with statistical descriptives. Ripley K-functions are used to examine the joint and marginal behavior of locations and fusion times. Semiautomatic detection and mapping of constitutive fusion sites reveals spatial and temporal clustering, but no dependency between the locations and times of fusion events. Our novel approach could be translated to other studies of membrane trafficking in health and diseases such as diabetes.

Exocyst is involved in polarized cell migration and cerebral cortical development

“…Since fusion events often clustered in small domains of the membrane, which we call hotspots, we tested whether Sec8-depletion causes a randomization of the locations of fusion events. We modeled the exocytic process as a spatial point process, ie, a random variable producing points at random locations representing the location of events (42). A homogenous Poisson process is a completely random point process, in which the number of events in nonoverlapping spatial regions is independent and the probability of an event is the same at all locations.…”

“…We used total internal ref lection f luorescent (TIRF) microscopy (38)(39)(40)(41)(42) in combination with RNAi against Sec8, the most frequently studied exocyst subunit. As a next step, we capitalized on the existence of a dominant negative mutant of the Exo70 exocyst subunit, which allowed us to test the importance of the exocyst directly in migrating cortical neurons during embryonic development.…”

A detection-based framework for the analysis of recycling in TIRF microscopy

“…Total Internal Reflexion Fluorescence [4] microscopy based on the properties of evanescent waves propagation at the interface between two media with sufficiently different refractive indexes, is certainly the method of choice. This widely used approach demonstrated that transient fluorescence concentrations can occur close to or at the plasma membrane, suggesting that they may correspond to membrane sites specialized in endocytosis or exocytosis [5]. Among diverse molecular behaviors reported by previous microscopy studies on this molecular model, plasma membrane concentration of fluorescently tagged proteins give rise to sudden spot appearance and/or disappearance.…”

A Guided Tour of Selected Image Processing and Analysis Methods for Fluorescence and Electron Microscopy

“…For instance, in [193], a point process modeling is proposed in order to characterize some properties of the endo/exocytosis process such as the presence of clusters from detecting spots. Generally, the manual detection of objects over a cluttered time-varying background is very tedious especially in 3D and subjective.…”

“…More specifically, some methodologies can be considered for the automatic detection of appearing and vanishing spots of fluorescently labelled clustered molecules in wide-field (WF) and TIRF microscopy images [193], [190]. The difficulty is to distinguish motions due to trafficking from the appearing and vanishing spots.…”