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
Kontrast fazowy 200 180 160 140 120 100 80 60 40 20 0 0 200 400 600 800 1000
DIC Nomarski 200 180 160 140 120 100 80 60 0 50 100 150 200 250 300 350 400
M. fluorescencyjna 160 140 120 100 80 60 40 20 0 0 50 100 150 200
CrossCorrelation Tracking
CrossCorrelation Tracking
Trajektorie 170.5764 85.2882 0-170.5764-85.2882 85.2882 170.5764-85.2882-170.5764
Trajektorie 170.5764 85.2882 0-170.5764-85.2882 0 85.2882 170.5764-85.2882-170.5764
Confocal microscope Leica TCS LSI - practical applications 1 Płatek R., 2 Korczyński J. and 1 Skup M. 1 Laboratory for Reinnervation Processes and 2 Laboratory 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?
Parametry Leica TCS LSI 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 Confocal Zoom Zoom range [x] Optical Zoom (Z16 APO) Laser type Number of lasers Laser options [nm] Excitation attenuation Macro-Objectives Working distance [mm] Micro-Objectives true confocal 1 yes 1 8 sequential 128, 256, 512,1024, 2048 8 or 12, switchable yes 430 750 1 ultra high dynamic PMT continuously variable 1x 16x 0.57 9.2x solid state max 4 405, 488, 532 or 561, 635 AOTF 1x, 2x, 5x 97/39/19 10x, 20x, 40x, 63x
Model doświadczalny
Miejsce iniekcji - transdukowane komórki Miejsce iniekcji - transdukowane komórki Lezja Aksony transdukowanych komórek Rdzeń kręgowy szczura preparat - R. Platek zdjęcie J.Korczynski + R. Platek Dane niepublikowane Instytut IBD Nenckiego
Porównanie czasowo-przestrzenne ekspresji EGFP w rdzeniach szczurów Rat TP 7.3 7 DPO DOOGŁOWOWO DOOGONOWO Rat TP 14.2 14 DPO DOOGŁOWOWO DOOGONOWO Rat TP 4.5 35 DPO DOOGŁOWOWO DOOGONOWO Rdzeń kręgowy szczura preparat - R. Platek zdjęcie J.Korczynski + R. Platek Dane niepublikowane Instytut IBD Nenckiego
Zakres świecenia komórek rdzeń (mm) (średnia +/- SEM) Zakres świecenia komórek rdzeń (mm) (średnia +/- SEM) Porównanie czasowo-przestrzenne ekspresji EGFP w rdzeniach szczurów 14 Sygnał EGFP - rdzeń świecenia" 14 Sygnał EGFP - pojedyncze komórki" 12 12 10 10 8 8 6 6 4 4 2 2 0 7 DPO 14 DPO 35 DPO 0 7 DPO 14 DPO 35 DPO
Rat 4.3 Rat 4.4 Doogonowa granica lezji DOOGONOWY Doogonowa granica lezji DOOGONOWY
Leica TCS LSI obrazowanie in vivo
Mózg mysi postmortem. preparat - R.Platek, zdjęcie - J.Korczynski + R.Platek Dane nieopublikowane Instytut IBD Nenckiego
Budowa opuszki węchowej opuszka węchowa kość komórka mitralna kłębuszek (glomerulus) sygnał przesyłany jest do wyższych struktur mózgowych sygnał przekazywany jest w kłębuszkach nabłonek węchowy węchowe komórki receptorowe węchowe komórki receptorowe są aktywowane i przesyłają pobudzenie
Budowa opuszki węchowej dorosła mysz Przekrój poprzeczny przez opuszkę węchową dorosłego samca myszy (szczep: C57BL/6j). Zdjęcie z mikroskopu konfokalnego - znakowanie jądrowe TOTO3 Niebieski warstwa kłębuszkowa z ciałami komórek około kłębuszkowych miejsce połączenia aksonów receptorów węchowych z apikalnymi dendrytami komórek mitralnych 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. Pseudokolory naniesione Photoshopem w celu pokazania 3 głównych warstw anatomicznych Źródło - Wikimedia Commons
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 prof. Małgorzata Skup Pracownia Mikroskopii Konfokalnej, Nencki Pracownia Procesów Reinerwacyjnych, Nencki Jarek Korczyński Pracownia Mikroskopii Konfokalnej, Nencki
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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
CYKL KOMÓRKOWY 4N 4N 2N 2N-4N
BADANIE CYKLU KOMÓRKOWEGO OPIERA SIĘ NA POMIARACH ILOŚCI DNA Z WYKORZYSTANIEM CYTOMETRU PRZEPŁYWOWEGO G1/G0 2N 4N G2/M apoptoza <2N S 2N< <4N poliploidy >4N
CYKL KOMÓRKOWY
Mitoza dziś RIEDER C.L., KHODJAKOV A., 2003 Science 300, 91-96
Mitoza dawniej W. Flemming, Zellsubstanz, kern und zelltheilung (Verlag Vogel, Leipzig, 1882). RIEDER C.L., KHODJAKOV A., 2003 Science 300, 91-96
Lata 20-te XX w
VIDEO-MIKROSKOPIA UMOŻLIWIA ŚLEDZENIE LOSÓW KOMÓRKI CZAS
VIDEO-MIKROSKOPIA WYMAGA KOMPROMISÓW Intensywność światła długość fali Czas Temperatura jakość obrazu żywotność komórek
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)
Matrix metallopeptidase 9 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. 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 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 P. Michaluk et al., J Cell Sci 124, 3369 (2011).
HYPOTHESIS
MMP-9-mediated spines morpholgy changes are integrins dependent MMP-9 integrins spine elongations
MMP-9 activate integrins 2. MMP-9 activates integrins directly 1. MMP-9 exposed integrin-activating epitope adapted from Dityatev et al., Nat Rev Neurosci (2010)
HYPOTHESIS VERIFICATION
Source of MMP-9 substrates: hippocampal tissue aammp-9 400ng/ml 37 o C, 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 -30 0 5 10 20 30
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
SpineMagick Local brightness variation: Halo correction: before correction after correction spines covered by dendrite halo Z-section Spine head anti-detachment: spine heads often separated; necks barely visible improved segmentation of spine neck Ruszczycki, Wlodarczyk,Kaczmarek
Kasia Zalewska Amanda Costa Marta Pyskaty Acknowledgement
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.