MOTION UNDER THE MICROSCOPE: MODERN TECHNIQUES FOR STUDYING CELL ADHESION AND MOTILITY

Paweł Pomorski

Laboratory of the Molecular Basis of Cell Motility

Nencki Institute of Experimental Biology, Warsaw, Poland

Ability to move is one of the fundamental functions of the living cells. It is due to the motility that organism develops, immune system can work, organs are able to regenerate and wound heal. In the same time motility studies are among methodologically most difficult ones. Biochemical processes underlying motility are notoriously unsynchronized and motile cells are usually not very numerous. Current paper reviews microscope techniques developed to solve those problems. We discuss basic measurements, parameterizing motility and substratum adhesion. Classical, structural microscopy used for motility studies are also sketched shortly. We describe also use of molecular probes for signaling studies in motile cells as well as we mention about microscopic experimental techniques, allowing experiments on single cells.

Ruch i adhezja komórek – metody mikroskopowe

Paweł Pomorski 18.06.2013

1. Obserwacja i parametryzacja ruchu komórek

2. Pomiar adhezji komórek 3. Zastosowanie w praktyce

doświadczalnej

ŚLEDZENIE KOMÓREK

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Confocal microscope Leica TCS LSI - practical applications 1Płatek R., 2Korczyński J. and 1Skup M.

1Laboratory for Reinnervation Processes and 2Laboratory for Confocal Microscopy

Nencki Institute of Experimental Biology, Warsaw, Poland.

Conventional confocal microscopy is dedicated mainly to proceed with thin tissue sections and cells seeded on a cover slip. The Leica TCS LSI macro confocal is the first super zoom microscope that combines all benefits of traditional confocal microscopy with large scale imaging of anesthetized, alive objects as well as unfixed/fixed objects post mortem. In our experiments we have tested Leica TCS LSI for scanning of (1) murine brains and spinal cords in vivo, and dissected in toto immediately post mortem, and (2) fixed rat spinal cords. We used transgenic mice expressing green fluorescent protein (GFP) under PLP promoter, to visualize oligodendrocytes, and rats with spinal cords transduced with AAV vector coding for enhanced GFP under mCMV promoter, to visualize neurons and glia. Super zoom confocal microscopy let us observe general distribution of GFP- expressing cells in whole organs as well as to focus on single cells and fibers. We were able to discriminate well between main morphological features of these cells. In murine brains we could visualize myelinated axons and oligodendrocytes, whereas in rat spinal cords the extent of eGFP expressing cells and their fibers traversing along entire spinal cord could be traced. The labeled objects could be visualized from the regions lying within a range of 100 μm from the surface of the brain/spinal cord. The details on the procedures applied, benefits and limitations of the method will be presented.

Mikroskop konfokalny Leica TCS LSI -

- przykłady zastosowania

Mikroskopia konfokalna - Praktyczny Kurs Badań In vivo

18-19.06.2013

Rafał Płatek; Pracownia Procesów Reinerwacyjnych

Mikroskopia konfokalna

?

Mikroskopia konfokalna

Scanner Method

Confocal channels

Scanner galvo (x,y)

Sequential scan

Channel multiplexing

Scan formats [pixel]

Image depth [bit]

Spectral detection

Spectral bandwidth [nm]

Detector

Detector type

true confocal

1

yes

1 – 8 sequential

128, 256, 512,1024, 2048

8 or 12, switchable

yes

430 – 750

1

ultra high dynamic PMT

Confocal Zoom

Zoom range [x]

Optical Zoom (Z16 APO)

continuously variable

1x – 16x

0.57 – 9.2x

Laser type

Number of lasers

Laser options [nm]

Excitation attenuation

solid state

max 4

405, 488, 532 or 561, 635

AOTF

Macro-Objectives

Working distance [mm]

Micro-Objectives

1x, 2x, 5x

97/39/19

10x, 20x, 40x, 63x

Mikroskopia konfokalna Parametry Leica TCS LSI

Model doświadczalny

Lezja

Miejsce iniekcji - transdukowane komórki

Rdzeń kręgowy szczura preparat - R. Platek

zdjęcie – J.Korczynski + R. Platek

Dane niepublikowane

Instytut IBD Nenckiego

Aksony transdukowanych komórek

Miejsce iniekcji - transdukowane komórki

DOOGŁOWOWO DOOGONOWO

7 DPO Rat TP 7.3

DOOGŁOWOWO DOOGONOWO

14 DPO Rat TP 14.2

DOOGŁOWOWO DOOGONOWO

35 DPO Rat TP 4.5

Porównanie czasowo-przestrzenne ekspresji EGFP w rdzeniach szczurów

Rdzeń kręgowy szczura preparat - R. Platek

zdjęcie – J.Korczynski + R. Platek

Dane niepublikowane

Instytut IBD Nenckiego

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Porównanie czasowo-przestrzenne ekspresji EGFP w rdzeniach szczurów

Rat 4.3

Doogonowa granica lezji DOOGONOWY

DOOGONOWY Doogonowa granica lezji

Rat 4.4

Leica TCS LSI – obrazowanie in vivo

Mózg mysi postmortem. preparat - R.Platek,

zdjęcie - J.Korczynski + R.Platek

Dane nieopublikowane

Instytut IBD Nenckiego

komórka mitralna

kłębuszek (glomerulus)

opuszka węchowa

kość

nabłonek węchowy

węchowe komórki receptorowe

węchowe komórki receptorowe są aktywowane i przesyłają pobudzenie

sygnał przekazywany jest w kłębuszkach

sygnał przesyłany jest do wyższych struktur mózgowych

Budowa opuszki węchowej

Źródło - Wikimedia Commons

Czerwony – warstwa

komórek mitralnych z

ciałami komórek

mitralnych i ziarnistych

w tej warstwie

Zielony – warstwa

komórek ziarnistych z

ciałami niedojrzałych,

migrujących

neuroblastów oraz

komórek ziarnistych.

Niebieski – warstwa

kłębuszkowa z ciałami komórek

około kłębuszkowych – miejsce

połączenia aksonów receptorów

węchowych z apikalnymi

dendrytami komórek mitralnych

Pseudokolory naniesione Photoshop-

em w celu pokazania 3 głównych

warstw anatomicznych

Budowa opuszki węchowej – dorosła mysz

Przekrój poprzeczny

przez opuszkę węchową

dorosłego samca myszy

(szczep: C57BL/6j).

Zdjęcie z mikroskopu

konfokalnego -

znakowanie jądrowe

TOTO3

Analiza całych struktur: kłębuszki opuszek węchowych u

myszy PLP

Mózg myszy PLP3 postmortem. preparat - R.Platek,

zdjęcia - J.Korczynski + R.Platek

Nieopublikowane dane

Instytut IBD Nenckiego

Podsumowanie i wnioski

Zalety mikroskopu konfokalnego TCS LSI:

• szybka ocena skuteczności zastosowanej metody doświadczalnej,

• szybkie pozyskanie wyniku w analizach nie wymagających bardzo

dużych powiększeń,

• analiza wyników na dużą skalę – skanowanie dużych powierzchni

(in vivo oraz in vitro) – trudne do przeprowadzenia na pojedynczych

skrawkach,

• duża elastyczność obrazowania – użycie obiektywów makro (1x, 2x i 5x)

w połączeniu z elektronicznym zoom-em.

Podziękowania:

Marian Kawczynski,

Kaw.aska, Zalesie Górne, Polska

• prof. Wanda Kłopocka Pracownia Mikroskopii Konfokalnej, Nencki

• Jarek Korczyński Pracownia Mikroskopii Konfokalnej, Nencki

• prof. Małgorzata Skup Pracownia Procesów Reinerwacyjnych, Nencki

Dziękuję za uwagę

Cell cycle analysis using time-lapse microscopy

Grażyna Mosieniak

Laboratory of Molecular Bases of Aging

Nencki Institute of Experimental Biology, Warsaw, Poland

Among many different techniques used in microscopy in order to visualize biological processes, time-laps microscopy gives unique opportunity to observe living cells on single cell level in time. One of the important processes that gain special interest is mitosis. Equal and undisturbed distribution of genetic material into two daughter cell that take place during mitosis is prerequisite of genome stability. In contrary, mitosis disturbances give rise to chromosomal instability, aneuploidy and cancer. Morphological changes that characterize mitotic cells enables to monitor cell division as well as tracking the fate of daughter cells. Moreover, transfection of the cells with vectors coding fluorescent proteins which expression changes during cell cycle make possible to follow the cell cycle progression of individual cell. One of the very interesting process that gain attention in cancer biology research is senescence of cancer cells. This process is induced during chemotherapy and thus it determine the outcome of it. Using video-microscopy we were able to show that aberrant mitosis could be the primary cause of senescence of cancer cells. Moreover video-microscopy enables to observe the fate of senescent cells and help to answer the question whether senescence of cancer cells lead to permanent growth arrest or is transient and could results in regrowth of cancer cells after therapy.

Cell cycle analysis using time-lapse microscopy

Grażyna Mosieniak

Laboratory of Molecular Bases of Aging

2N

2N-4N

4N

4N

CYKL KOMÓRKOWY

G1/G0 G2/M 2N 4N

2N< <4N <2N

S

apoptoza

>4N

poliploidy

BADANIE CYKLU KOMÓRKOWEGO OPIERA SIĘ NA POMIARACH ILOŚCI DNA Z WYKORZYSTANIEM CYTOMETRU PRZEPŁYWOWEGO

CYKL KOMÓRKOWY

RIEDER C.L., KHODJAKOV A., 2003 Science 300, 91-96

Mitoza dziś …

RIEDER C.L., KHODJAKOV A., 2003 Science 300, 91-96

Mitoza dawniej … W. Flemming, Zellsubstanz, kern und zelltheilung (Verlag Vogel, Leipzig, 1882).

Lata 20-te XX w

CZAS

VIDEO-MIKROSKOPIA UMOŻLIWIA ŚLEDZENIE LOSÓW KOMÓRKI

jakość obrazu

żywotność komórek

VIDEO-MIKROSKOPIA WYMAGA KOMPROMISÓW

• Intensywność światła • długość fali • Czas • Temperatura

Live imaging of influence of MMP-9 enzymatic activity on spine morphology through integrins. Izabela Rutkowska-Włodarczyk Laboratory of Neurobiology Nencki Institute of Experimental Biology, Warsaw, Poland

Synaptic plasticity can be defined as a re-organization comprising both, alterations at

the morphological level and changes at the functional level of dendritic spines, carrying

postsynaptic domains of excitatory synapses. It has been reported that motor learning leads to

rapid formation of dendritic spines (spinogenesis) in the motor cortex mice (Xu et al., 2009; Yang

et al., 2009). Changes in spine dynamics are region- and learning-specific, indicating that motor

learning causes synaptic reorganization in the corresponding motor cortex (Xu et al., 2009) and

synaptic connections are not only capable of undergoing rapid changes in response to new

experience but also can serve as substrates for long-term information storage (Yang et al.,

2009). This is why it became important to effectively quantify changes in dendritic spine

morphology. However, the results of this quantitative analysis can be largely influenced by the

the diversity in dendritic spine population. The detection of differences in spine morphology

between control and test group is often compromised by the number of dendritic spines taken

for analysis. For example, to have statistically significant detection of hidden morphological

differences between control and test groups in terms of spine head-width, length and area in

which the values of each morphometric variable under investigation grew in a 20% rate there

are required 12, 21 and 24 cells respectively with around 30 spines/cell. Simulation of changes

occurring in a subpopulation of spines reveal the strong dependence of detectability on the

statistical approach applied (Ruszczycki et al. 2012). This is way the best method to track spine

morphology changes is live imaging.

Live imaging of influence of MMP-9 enzymatic activity on spine morphology

through integrins.

Izabela Rutkowska-Wlodarczyk

Laboratory of Neurobiology

The Nencki Institute of Experimental Biology

OVERVIEW OF THE PROJECT

Changes in dendritic spine morphology underlie learning processes

(Xu et al., Nature,2009; Yang et al., Nature, 2009)

extracellurally operating protease that is expressed by the neurons and released in response to enhanced neuronal activity Michaluk et al., J. Biol. Chem., 2007

plays a key role in synaptic plasticity associated with memory and learning processes; Nagy et al, J. Neurosci., 2006;

Okulski et al., Biol. Psychiatry, 2007

was found to be present in a subset of dendritic spines bearing asymmetric synapses; Wilczynski et al., J. Cell Biol., 2008.

Matrix metallopeptidase 9

Dziembowska&Wlodarczyk, Int. J. Biochem. Cell Biol, (2012)

Integrins:

• play a role in the synaptic structural changes associated with LTP (Lee et al., 1980).

• stabilizing LTP (Staubli et al., 1990)

Dendritic spine elongation and new filopodia formation are induced by

integrin activating peptide.

Enzymatic activity of MMP-9 causes elongation and thinning of dendritic

spines

P. Michaluk et al., J Cell Sci 124, 3369 (2011). Shi &Ethell, J Neurosci 26, 1813 (2006).

P. Michaluk et al., J Cell Sci 124, 3369 (2011).

MMP-9 dependent spine morphology changes are integrin b1 dependent

HYPOTHESIS

MMP-9 spine elongations

integrins

MMP-9-mediated spines morpholgy changes are integrins dependent

MMP-9 activate integrins

1. MMP-9 exposed

integrin-activating

epitope

2. MMP-9 activates

integrins directly

adapted from Dityatev et al., Nat Rev Neurosci (2010)

HYPOTHESIS VERIFICATION

Source of MMP-9 substrates: hippocampal tissue

aaMMP-9 400ng/ml

37oC, 1h

MMP-9 Integrin conneXtion

Identification of MMP-9 substrates which serve

as integrin activators

MIX

GFP-transfected 21 DIV hipocampal culture

Zeiss LSM780

‘Live’ experiments-protocol

inhibitor MIX

0 -30 10 20 30 5

‘Live’ experiments allow to track changes in the

spines morphology

Filopodia Thin Mushroom Stubby

Quantitative analysis of spines morphology was

done by the use of Spine Magick

Ruszczycki, Wlodarczyk, Kaczmarek

Z-section

Halo correction:

Spine head anti-detachment:

spine heads often separated; necks barely visible

spines covered by dendrite halo

improved segmentation of spine neck

before correction after correction

Local brightness variation:

Ruszczycki, Wlodarczyk,Kaczmarek

SpineMagick

Acknowledgement

Kasia Zalewska Amanda Costa Marta Pyskaty

Thank you for your attention

The application of the FRAP technique and live cell imaging to investigate an impact of HAX-1 protein on the formation and movement of P-bodies in HeLa cell line. Ewa A. Grzybowska Cancer Center Institute, Warsaw, Poland HAX-1 is a multifunctional protein involved in several key processes like apoptosis, cell migration and regulation of calcium homeostasis, but its mechanisms of action remain unknown. HAX-1 is also known to bind to the 3'UTR of at least two mRNA targets. Our research indicate that HAX-1 co-localizes with P-body marker, decapping protein 1 (Dcp1). Using Fluorescence Recovery After Photobleaching (FRAP) technique we demonstrated that HAX-1 dynamically interchanges between P-body and the cytoplasm, but it is also bound in P-body to significant extent. Concurrently, using live-cell imaging, we showed that HAX-1 is not responsible for the immobilization of P-bodies on actin filaments.