3D HDO-CLEM: cellular compartment analysis by correlative light-electron microscopy on cryosections.

Fundamental to obtaining a depth-understanding of the function and structure of cells is the ability to study and correlate their molecular topography with the ultrastructural morphology, for example, to visualize the position of a given protein relative to a given cell compartment and its morphology. Standard fluorescence light microscopy (FLM) relies on simple sample preparations, and localizes proteins in living or fixed cells with a resolution in the range of few hundred nanometers, allowing large field of view. However, FLM is unable to visualize the unlabeled cellular context. On the other hand, electron microscopy (EM) techniques reveal protein topology with the resolution in a range of a few tens of nanometer, retains the cellular context, but can only be applied on a limited field of view. Therefore, both approaches present shortcomings, in terms of field of view, statistical output, resolution, sample preparation, and context analysis, that can likely complement each other. To bridge the gap between FLM imaging and EM, several laboratories have developed methods for correlative light-electron microscopy (CLEM). In a nutshell, CLEM enables one to investigate the same exact region of interest utilizing the two microscope platforms, and thereby virtually combine their capabilities. We describe a protocol based on immunolabeling of Tokuyasu cryosections that allows correlation of LM and EM images with excellent preservation of cellular ultrastructure. We will refer to this method as high-data-output CLEM (HDO-CLEM). The major benefits of HDO-CLEM are the possibility to (1) correlate several hundreds of events at the same time, (2) perform three-dimensional (3D) correlation, (3) immunolabel both endogenous and recombinantly tagged proteins at the same time, and (4) combine the high data analysis capability of FLM with the high precision of transmission EM in a CLEM hybrid morphometric analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D FLM reconstruction and set the basis for a hybrid light/EM morphometry approach

ABCD 2015, Biennial Congress of the Italian Association of cell biology and differentiation

HSP90 inhibition induces ErbB2 cleavage and internalization simultaneoulsy with alterations in endosomal trafficking/maturation and autophag

ABCD Meeting \u201cOrganelle Biogenesis and Signal Transduction\u201d

An ubiquitin-proteasome-endocytic pathway but not autophagy orchestrates ErbB2 internalization and cleavage in HSP90-inhibited breast cancer cell

71\ub0 CONGRESSO NAZIONALE SIAI, Societ\ue0 di Anatomia e Istologia, Taormina, 20 - 22 Settembre 2017

The receptor tyrosine kinase ERBB2 interacts with HSP90 and is overexpressed in aggressive breast cancers. Therapeutic HSP90 inhibitors, i.e. Geldanamycin (GA), target ERBB2 to degradation. We have previously shown that HSP90 is responsible for the missorting of recycling ERBB2 to degradation compartments. In this study, we used biochemical, immunofluorescence and electron microscopy techniques to demonstrate that in SKBR3 human breast cancer cells, GA strongly induces polyubiquitination and internalization of the full-length p185-ERBB2, and promotes its cleavage, with the formation of a p116-ERBB2 form in EEA1-positive endosomes (EE). p116-ERBB2 corresponds to a non-ubiquitinated, signaling-impaired, membrane-bound fragment, which is readily sorted to lysosomes and degraded. To define the sequence of events leading to p116-ERBB2 degradation, we first blocked the EE maturation/trafficking to late endosomes/lysosomes with wortmannin, and found an increase in GA-dependent formation of p116-ERBB2; we then inhibited the proteasome activity with MG-132 or lactacystin, and observed an efficient block of p185-ERBB2 cleavage, and its accumulation in EE, suggesting that p185-ERBB2 polyubiquitination is necessary for proteasome-dependent p116-ERBB2 generation occurring in EE. As polyubiquitination has also been implicated in autophagy-mediated degradation of ERBB2 under different experimental conditions, we exploited this possibility and demonstrate that GA strongly inhibits early autophagy, and reduces the levels of the autophagy markers atg5-12 and LC3-II, irrespective of GA-induced ERBB2 polyubiquitination, ruling out a GA-dependent autophagic degradation of ERBB2. In conclusion, we propose that HSP90 inhibition fosters ERBB2 polyubiquitination and proteasome-dependent generation of a non-ubiquitinated and inactive p116-ERBB2 form in EE, which is trafficked from altered EE to lysosomes

Trimming the resolution gap in the study of molecular and cellular events by means of High Data Output and automated three-dimensional Correlative Light-Electron Microscopy approach

Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in molecular and cellular biophysics. Here we report on a new high data output three-dimensional (3D) CLEM method based on the use of cryosections (Vicidomini et al.,Traffic, 2008). We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibility to perform 3D correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. The relevance of the presented approach is further enhanced by two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques

A novel approach for correlative light electron microscopy analysis.

Correlative light and electron microscopy (CLEM) is a multimodal technique of increasing utilization in functional, biochemical, and molecular biology. CLEM attempts to combine multidimensional information from the complementary fluorescence light microscopy (FLM) and electron microscopy (EM) techniques to bridge the various resolution gaps. Within this approach the very same cell/structure/event observed at level can be analyzed as well by FLM and EM. Unfortunately, these Studies turned out to be extremely time consuming and are not suitable for statistical relevant data. Here, we describe a new CLEM method based on a robust specimen preparation protocol, optimized for cryosections (Tokuyasu method) and on an innovative image processing toolbox for a novel type of multimodal analysis. Main advantages obtained Using the proposed CLEM method are: (1) hundred times more cells/structures/events that can be correlated in each single microscopy session; (2) three-dimensional correlation between FLM and EM, obtained by means of ribbons of serial cryosections and electron tomography microscopy (ETM); (3) high rate of success for each CLEM experiment, obtained implementing protection of samples from physical damage and from loss of fluorescence; (4) compatibility with the classical immunogold and immunofluorescence labeling techniques. This method has been successfully validated for the correlative analysis of Russel Bodies subcellular compartments. Microsc. Res. Tech. 73:215-224, 2010. (c) 2009 Wiley-Liss. Inc