Základní metody studia buněk

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1 Základní metody studia buněk
extrakce a frakcionace pozorování ve světelném a elektronovém mikroskopu barvení buněčných komponent a fluorescenční mikroskopie

2 Součásti buněk lze oddělit centrifugací

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7 LM vs. TEM

8 Bright-field light microscope

9 Types of LM: A) Bright field B) Phase contrast

10 Types of LM: C) Diff. Interference Contrast
D) Dark field

11 Bright field microscopy of stained section of kidney tubules

12 Indirect Immunocytochemistry

13 Stain for DNA. What is fluorescence? Energy of light is inversely proportional to the wavelength. For example, violet light has higher energy than red light. Fluorescent molecules are special molecules that absorb incident light of a particular wavelength then emit light of a longer wavelength. The absorption of light causes electrons in the molecule to become highly energetic. When the highly energetic electrons return to their original state, they emit light. Wave length of incident light must be shorter than the wavelength of emitted light since some energy is unavoidably lost as heat.

14 Fluorescence Microscope

15 Fluorescence image of a cell in mitosis labeled w 3 tags:
Green: fluorescein-labeled anti-tubulin (MTS) Red: anti- centromere protein Blue: DAPI (DNA)

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17 Images of Conventional (A) vs Confocal (B) Fluorescence
(Drosophila embryo stained w fluorescent probe against actin)

18 no FBS MBF1 DAPI Actin control Actinomycin D Doxorubicin

19 Mnohobuněčnost a diferenciace
určení buněčného osudu diferenciální genová aktivita modelové organismy pro vývojovou biologii

20 Buněčný cyklus je řízený sled událostí vedoucí ke zdvojení (dělení) buněk
jediný chromosom

21 Diferenciace

22 Typy buněk ve střevním epitelu

23 CELL FATES Gamety Progenitor cells Differentiated cells Stem cells
determination/commitment differentiation Gamety Progenitor cells Differentiated cells meiosa Zygota Stem cells Differentiated cells quiescence senescence obnova Differentiated cells apoptosa neomezený růst Mrtvé buňky Nádorové buňky

24 Rakovina pochází z proliferujících buněk
Tvorba diferenciovaných krevních buněk z hematopoetických kmenových buněk kostní dřeně

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26 Různé typy buněk exprimují různé proteiny

27 Modelové organismy moucha octomilka Drosophila melanogaster
hlístice háďátko Caenorhabditis elegans žába (Xenopus), kuře a myš

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32 Antennae conversion to legs!

33 Haltere to wing transformation
Homeotic Mutations; The conversion of one body part into another Haltere to wing transformation

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36 Caenorhabditis elegans
Less than 1000 cells in adult. Self-fertilizing hermaphrodite Short generation time Sydney Brenner:

37 The nematode worm C. elegans

38 1090 cells, 131 die, leaving 959 in adult.
Idea: It might be possible to trace the developmental lineage of every single cell in the adult worm Zygote John Sulston 1090 cells, 131 die, leaving 959 in adult.

39 Early cell lineage in C. elegans

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41 “Without additional information, no amount of gazing at genome sequences will the functions of genes.” Alberts and colleagues: Molecular Biology of the Cell, 4th edition, page 26.