Correlative microscopy ALBERTO LUINI INSTITUTE OF PROTEIN BIOCHEMISTRY, CNR ROMAN POLISHCHUCK TIGEM

NAPLES

THE IMAGING FACILITY AT THE CASTELLINO CAMPUS The Castellino Campus comprises a number of Institutes of the National Research Council (CNR), among which the main ones are the IBP and the Institute of Genetics and Biophysics (IGB). Other Castellino Institutes operate in the fields of photonics (IMM), informatics and computational modeling, and imaging (IAC, ICAR). The Campus also hosts a large Telethon research Institute, the Telethon Institute for Genetics and Medicine (TIGEM). The IBP, IGB and TIGEM each possess an imaging facility. The IMM, IAC and ICAR develop technology in the field of photonics and computational image analysis, and collaborate with the imaging facilities. In addition, the IBP hosts the Telethon service Facility for advanced electron microscopy, tomography and correlative microscopy. All of these facilities are managed in a coordinated manner that is regulated by formal agreements among the Institutes, and are open to all of the members of the Castellino Campus. The Telethon facility at the IBP has extensive experience in access, as it has offered service and assistance to dozens of Telethon laboratories for a number of years. In many cases the support provided by facility allowed Telethon-funded scientist to publish their data in top journals (see selected publications below). he criteria and practice of access developed by the Telethon EM facility at IBP will be used to run the EuroBioImaging proof of concept study (PCS).

The instrumentation available at the integrated Castellino facilities include Light microscopy - Confocal microscope Zeiss 710 - Confocal microscope Leica SP5 - Confocal microscope Leica SP2 - Fully motorized Leica DMI6000 light microscope equipped with incubation system for live cell imaging - 3 Leica DMI6000 light microscopes equipped with image acquisition and analysis systems - 2 Zeiss Axiophot microscopes equipped with image acquisition and analysis systems Electron Microscopy - FEI TEcnai G2 Spirit BioTWIN for EM tomography - FEI TEcnai G2 Spirit BioTWIN for EM tomography (accessible in the CNR Institute ICTB) - JEOL JEM-1011 electron microscope - Leica Ultracut UCT ultramicrotome - Leica EM FC7 ultramicrotome - Leica EM TP automated tissue processor Other equipment - The accessory equipment comprises centrifuges, ovens, shakers and steromicroscopes for specimen preparation - Eppendorf microinjection station - 5 off line PC stations for EM and light microscopy image analysis

PERSONNEL EXPANSION

SERVICE The Telethon Electron Microscopy Core Facility (TeEMCoF) The Main Goal To help Telethon funded studies of genetic disease with electron microscopy

Services

Short list of the diseases studied by TeEMCoF  Juvenile nephropathy

Over 30 projects in 2005-2010

 Ocular albinism  Juvenile hemochromatosis

Milano

Padova

 Prion protein disease

 CLEM

 Lysosomal storage diseases

 EM tomography

 Muscular dystrophy

 Immuno-EM

 Diabetes

 Routine EM

 Neurodystrophy

 Morphometry

 Optic atrothy

 Equipment use  Training About 40 papers published in 2005-2010 comprising top journals (Cell, Nature, etc.)

Siena

Perugia Chieti

Roma

Bari Napoli

UNIQUENESS

Euro-BioImaging. European Biomedical Imaging Infrastructure- from Molecule to Patient. A project on the European Roadmap for Research Infrastructures under coordination of EMBL and EIBIR ADVANCED LIGHT MICROSCOPY NODES Advanced light microscopy is instrumental to reach the ultimate goal of biological imaging, to visualise single biomolecules and their functions and interactions within the context of live biological systems. The specific nodes will provide the following key technologies:

Superresolution light microscopy This node will provide access to methods that improve the spatial (and also temporal) resolution of light microscopy imaging with an emphasis on technologies applicable for biological applications and in live specimens. Key technologies will include stimulated emission depletion (STED), photoactivation localization microscopy (PALM) as well as the use of structured illumination.

Functional imaging of live cells This node will provide access to methods that visualise molecular function in live cells. Key technologies will include fluorescence lifetime imaging (FLIM), fluorescence (cross) correlation spectroscopy (FC[C]S), photoactivation and photobleaching (PA, FRAP), single molecule imaging, and novel fluorescent reporters of biochemical reactions.

Correlative light and electron microscopy In this node it will be possible to combine dynamic functional assays in live cells directly with high resolution 3D morphology at molecular resolution by EM (cryo) tomography. This node will be intimately linked to EM activities in the ESFRI initiative INSTRUCT.

High throughput microscopy for systems biology This node will contribute to systems biology and rational drug development by providing access to automation and high throughput in advanced light microscopy methods including ultra high content screening of genome level systematic perturbations of biological systems such as RNA interference overexpression or small molecule screening.

CORRELATIVE MICROSCOPY

INTEGRATED MICROSCOPY Attempts to apply different microscopy approaches to the very same object of interest to integrate information about the dynamics, fine structure and composition of that object

VIDEO – EM VIDEO - IF VIDEO - IF - EM

A FORM OF SUPERRESOLUTION VIDEO MICROSCOPY

Principle Characterize dynamics by video microscopy Switch to electron microscopy Characterizing a dynamic process in live cells Choosing a particular structure (stage) of interest in the process being studied Taking two pictures of the same structure, one at the LM, and the other at the EM level. Integrating the information Advantages Magnification Structure (reference space) Disadvantages Fixation Probes Retracing

Correlative video-electron microscopy Immunoperoxidase

Immunoperoxidase EM Polishchuk et al, J. Cell Biol. (2000)

3-D tomography

3-D reconstruction

A COMPLEX INVOLVED IN CARRIER FORMATION AND FISSION BY SERIAL BLEACHING AND LONG IMMUNO-FRET

COMBINING VIDEO MICROSCOPY WITH EM TOMOGRAPHY CRYO-FIXATION IMMUNO-GOLD LABELING AND TOMOGRAPHY DETECTION OF MOLECULAR COMPONENTS AND OF MOLECULAR COMPLEXES IN OBJECTS OF INTEREST

A COMPLEX INVOLVED IN CARRIER FORMATION AND FISSION BY SERIAL BLEACHING AND LONG IMMUNO-FRET

FRET distance (Å) from D to A

D A protein complex

Förster Resonance Energy Transfer

THE IDEAL CONFIGURATION FOR LONG RANGE FRET MULTIPLE ACCEPTORS, LONG FRET DISTANCE

distance (Å) from D to A multiple acceptors

D A single donor

Molecular complex

Single donor molecule allows an higher efficiency of energy transfer for a cumulative effect and eliminates the problem of homotransfer

Increasing FRET efficacy

DONOR-ACCEPTOR distance (Å)

DETECTION OF MOLECULAR COMPLEXES BY FRET SERIAL LABELING – ACQUISITION – BLEACHING

DIFFICULTY: THE SIZE OF THE COMPLEXES

A COMPLEX INVOLVED IN CARRIER FORMATION AND FISSION

CORRELATIVE MICROSCOPY INTEGRATES INFORMATION ON : DYNAMICS ULTRASTRUCTURE MOLECULAR COMPOSITION ASSEMBLY OF MOLECULAR MACHINERY

Multiple labelling of budding post-Golgi carriers by serial bleaching VSVG-GFP VSVG-GFP Release from 20°C block

FAPP-2

CERT

PI4K

A COMPLEX INVOLVED IN CARRIER FORMATION AND FISSION BY SERIAL BLEACHING AND LONG IMMUNO-FRET

SERIAL BLEACHING OF PRIMARY ANTIBODIES

Schubert W, 2006

Protein complexes have different sizes, number of components (often many) and stability over time

a proteasome, a large molecular complex

clathrin, a transient protein assembly

A few moments of time-lapse video are enough to resolve an Issue that years of microscopy on fixed cells have failed to settle- Hugh R.B. Pelham (Nature. 1997)

Unfortunately, light microscopy cannot achieve a sufficiently high resolution, so spectacular through it is, GFP technology has its limits-Hugh R.B. Pelham (Nature. 1997)

The development of correlative microscopy can be somewhat arbitrarily divided divided into two stages 1) An early stage from 1960 or earlier. The goal is generally to look at the same field by both light (imuno-fluorescence) and electron microscopy, to exploit the advantages of the two techniques: the broad field of view of light and the resolution of electron microscopy. 2) A recent stage from 2000 onward. Correlative microscopy as the first kind of GFP-based super-resolution video microscopy with some disadvantages and a few substantial advantages over other superresolution video techniques that are being developed today

1986 Hayat MA. Correlative microscopy in Biology instrumentations and methods. Academic Press. 1987

1995

TRANSPORT CARRIERS What kind of fine structure do they have? With what organelles do they interact?

GFP-BASED CORRELATIVE LIGHT-ELECTRON MYCROSCOPY (CLEM): EXPERIMENTAL PROCEDURE 1. DNA transfection of cells grown on CELLocate coverslip.

2. GFP-based time-lapse confocal microscopy and fixation of the cells.

3. Immunoperoxidase or gold labelling and embedding in resin.

4. Cutting of serial sections and identification of structure of interest at EM level.

resin block

knife section

Collection of serial sections on the slot grids and their analysis at the electron microscope Perfect Loop

Analysis of serial sections

Serial

sections

Slot grid

3D reconstruction

Post-Golgi transport carriers in living cell VSVG-GFP Plasma membrane

Golgi

ER

Questions to address Plasma membrane 3

• How are post-Golgi carriers organized during different stages of their life-cycle? 1. Formation 2. Transition through cytosol 3. Fusion

2 1

Golgi

VSVG is a widely used protein to study membrane transport

VSVG-GFP

VSVG protein is a ts045 mutant strain of vesicular stomatitis virus

Lumen

40°C-VSVG is in the ER Cytosol

20°C-VSVG is in the Golgi (TGN) 32°C-VSVG is moved out of the ER or the Golgi

Correlative light-electron microscopy of VSVG-GFP labelled post-Golgi membrane carrier VSVG-GFP

Polishchuk R.S. et al. ( 2000, JCB)

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Formation of post-Golgi carriers Plasma membrane

Golgi

Plasma membrane

Golgi

Formation of post-Golgi carriers VSVG-GFP

Ultrastructure of exit site of post-Golgi carrier VSVG-GFP

Polishchuk E.V. et al. ( 2003, MBC)

Ultrastructure of post-Golgi carrier formation site

Polishchuk E.V. et al. ( 2003, MBC)

Ultrastructure of post-Golgi carrier formation site 1

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3

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Polishchuk E.V. et al. ( 2003, MBC)

Ultrastructure of post-Golgi carrier formation site 1

2

3

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Polishchuk E.V. et al. ( 2003, MBC)

Visualization of post-Golgi carrier fusion with the plasma membrane by transmission EM VSVG-GFP

VSVG-GFP

Anti-VSVG HRP

Anti-VSVG HRP Polishchuk R.S. et al. ( 2000, JCB)

Visualization of post-Golgi carrier fusion with plasma membrane by scanning EM

scanning EM confocal microscopy

Anti-VSVG gold

VSVG-GFP

Polishchuk R.S. et al. ( 2000, JCB)

Constitutive transport from the Golgi to the plasma membrane Plasma membrane

Golgi

Polishchuk R.S. et al. ( 2000, JCB)

Transport of cargo proteins from the Golgi to the apical and basolateral surfaces Apical surface

QUESTIONS TO ADDRESS • Is ultrastructure of apical and basolateral carriers different?

• Are apical and basolateral cargoes ever packed into the same post-Golgi carrier? Golgi

•If so, how do they distribute within these structures?

ER

Basolateral surface

Fluorescent proteins with apical and basolateral sorting signals show polarized distribution in MDCK cells Apical marker

GPI-GFP

Lumen

Basolateral marker

GPI-GFP

VSVG-GFP

Anti-occludin

Anti-occludin

Cytosol

VSVG-GFP XY

XY

XZ

XZ

Lumen

Cytosol

Polishchuk R.S. et al. ( 2004, NCB)

Dynamics of Golgi-to-plasma membrane transport of apical and basolateral markers in living cells merge

VSVG-CFP

GPI-YFP

Polishchuk R.S. et al. ( 2004, NCB)

Ultrastructure of post-Golgi carriers containing both apical and basolateral markers 2

1

merge

VSVG-CFP

GPI-YFP Polishchuk R.S. et al. ( 2004, NCB)

Ultrastructure of Golgi-to-plasma carriers in cell expressing apical and basolateral markers 1

1

2 2 Polishchuk R.S. et al. ( 2004, NCB)

Intracellular membrane transport Plasma membrane Secretory granules

Endosomes Lysosomes

Clathrin AP1 GGAs MPR Golgi

ER

3

2

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trans-Golgi network (TGN)

Structure of Golgi-to-endosome carriers

GFP view

EM view

- pleiomorphic carriers

- clathrin coated vesicles

- frequently tubules

- or vesicular clusters

- tubular or vesicular clusters

CLEM as a tool to characterize newly-formed endosomal transport carriers Constructs CD-MPR-GFP-Cation-Dependent Mannose 6-Phosphate Receptor-GFP GGA1-GFP-Golgi-localized Gamma-ear -containing ARF-binding protein1-GFP Clathrin light chain-GFP

GGA-GFP positive carriers in living cells

Polishchuk R.S. et al. ( 2006, Traffic)

CLEM of GGA-GFP positive carriers

GGA-GFP TRITC-dextran

Polishchuk R.S. et al. ( 2006, Traffic)

CLEM of GGA-GFP positive carriers

GGA-GFP TRITC-dextran

Polishchuk R.S. et al. ( 2006, Traffic)

Clathrin-coat domains are always present on GGA-GFP positive carriers

Polishchuk R.S. et al. ( 2006, Traffic)

Three-dimensional organization of GGA-GFP positive carriers

Polishchuk R.S. et al. ( 2006, Traffic)

Variability of shapes through GGA-GFP and MPR-GFP positive TC populations

vesicle

tubule

ovoid

grape-like Polishchuk R.S. et al. ( 2006, Traffic)

SUMMARY • TGN-to-endosome TCs exhibit various morphology ranging from vesicle-like to complex grape-like • Frequently such TCs only partially covered with clathrin • GGA adaptors are not restricted to the clathrin-coated domains

Overcoming resolution limits in light microscopy

4Pi-microscopy: Engineering the Point Spread Function (PSF) 2Pi angle

2Pi angle becomes a 4Pi angle

500 nm

100 nm

200 nm

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PSF nm

A laser-scanning microscopy with a resolution of

nm

80-100 nm along

the xy axes and even the Z axis! (Hell, Nature Biotechnology, 2003)

Confocal

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4Pi z

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y Perinetti G. et al. ( 2009, Traffic)

4Pi-CLEM Technique Confocal microscopy

4Pi-microscopy

DNA transfection

Electron microscopy EM processing

Confocal Recording

3D Reconstruction

4Pi Recording

Deconvolution

3D Reconstruction Comparison

Zero-Crossing

EM serial recording

3D Reconstruction Comparison

Threshold Threshold

Serial Sectioning

Zero-Crossing

Mini-stack visualization under the microscopes Non –Confocal Trasmission

Confocal

Non Confocal Fluorescence

TEM

4Pi

Mini-stack identification under the TEM recording

MS05

MS05 MS04

MS04 MS03

MS02

MS01

MS03

MS02

MS01

Perinetti G. et al. ( 2009, Traffic)

TEM serial recording and Imod labelling

4Pi-EM overlap Threshold 20%

z

Zero-Crossing

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4Pi-EM overlap

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Perspectives

• Combination with advanced light microscopy methods • Development of the new light-electron microscopy probes • Combination with proteomics approaches

Acknowledgements Mario Negri Sud Institute and TIGEM Alexander Mironov

Albeto Luini Elena Polishchuk Alexander Mironov Jr. Giuseppe Perinetti Alexander Spaar NICHD (NIH) Bethesda Jennifer Lippincott-Schwartz

Juan Bonifacino MPI (Goettingen) Tobias Muller Alexander Egner Stefan Hell

The Monte Carlo analysis: a simulation of the system distance (Å) from D to A multiple acceptors

D single donor

A protein complex

A typical experiment: complexes composition should be determined using standard biochemical techniques

video-microscopy to survey the cellular behaviour upon the stimulating event

cells are fixed at the time of interest, then sequential steps of:

immunolabeling with a donor and various acceptors long range FRET assessments  selective bleaching of acceptors

staining another protein with new acceptors NOR TIME NEITHER SPACE INFORMATIVE

TIME AND SPACE

ONLY SPACE

1994