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Lipidne kapljice (LK) so nedavno priznani organeli, ki so poleg pomembne vloge pri shranjevanju lipidov za proizvodnjo energije vpleteni tudi v uravnavanje celičnega stresa. LK se v celici lahko hitro sintetizirajo in razgrajujejo kot odziv na različne okoljske dejavnike in energetsko stanje celic. Rezultati raziskav v zadnjih letih so pokazali, da so LK v zapletenem odnosu z avtofagijo, ki se aktivira ob določenih oblikah celičnega stresa in vključuje lizosomsko razgradnjo citoplazemskih organelov ali citosolnih komponent v celici. Nekatere študije razkrivajo, da avtofagija lahko zagotavlja maščobne kisline za biogenezo LK, druge pa, da sodeluje tudi pri razgradnji LK v procesu selektivne oblike avtofagije, imenovane »lipofagija«. Prav tako so pokazali, da so LK lahko vir lipidov za izgradnjo avtofagosomskih membran in s tem omogočijo začetek procesa avtofagije. Ni še povsem jasno, kako sta ta dva zapletena mehanizma med seboj povezana in katere molekularne poti so odgovorne za posamezen izid. To je še posebej pomembno pri rakavih celicah, ki so pogosto izpostavljene neravnovesju hranil v tumorskem mikrookolju in lahko uporabljajo tako avtofagijo kot LK za zaščito pred stresom. V tej magistrski nalogi smo raziskali osnovne povezave med avtofagijo/lipofagijo in LK v visoko invazivnih celicah raka dojke, izpostavljenim pomanjkanju hranil. Pri tem smo optimizirali nekatere že ustaljene in vpeljali nove metode za vizualizacijo in kvantifikacijo interakcij med avtofagosomskimi strukturami in LK s konfokalno mikroskopsko analizo živih celic. Ugotovili smo, da se avtofagija aktivira ne le pri intenzivnem stradanju v odsotnosti aminokislin in seruma, temveč tudi pri blažjem stradanju v gojišču brez seruma. Pokazali smo, da inhibicija poznih stopenj avtofagije z bafilomicinom A1 ali klorokinom vodi do zmanjšanja vsebnosti LK pri intenzivnem stradanju, kar kaže na možnost, da avtofagija sodeluje pri biogenezi LK pri teh pogojih. Po drugi strani pa smo s poskusi konfokalne mikroskopije na živih celicah pokazali, da se avtofagosomske in lizosomske strukture v večji meri kolokalizirajo z LK med blagim, ne pa tudi med intenzivnim stradanjem, kar nakazuje na aktivacijo razgradnje LK z lipofagijo le pri blažjem stradanju. Pomanjkanje seruma je namreč privedlo do izrazite kolokalizacije med LK in fluorescenčnima proteinskima označevalcema za avtofagosome (mRFP-LC3) in lizosome (LAMP1-RFP). Naši poskusi prav tako kažejo, da vsaj v prvih 24 h stradanja zaviranje avtofagije z inhibitorji nima bistvenega vpliva na preživetje celic raka dojke. Če povzamemo, naše raziskovalno delo kaže na dvojno, od vrste stradanja odvisno vlogo avtofagije pri sintezi in razgradnji LK v celicah raka dojke in razkriva nova spoznanja, ki bodo prispevala k razlagi dinamike stresnega odgovora rakavih celic. Lipid droplets (LDs) are recently recognized organelles that have an essential role in lipid storage for energy production, but are also involved in the regulation of cellular stress. LDs can rapidly form or break down in response to various environmental factors and cellular energy levels. Recent research has shown that LDs are also engaged in a complex relationship with autophagy, a major cellular stress response mechanism involving lysosomal degradation of cytoplasmic organelles and various cytosolic material. Some studies reveal that autophagy can provide fatty acids for LD biogenesis, while other suggest that it is also involved in the breakdown of LDs via a selective form of autophagy called “lipophagy”. It has also been shown that LDs can be sources of lipids for autophagosome membrane assembly and thus enable the initiation of autophagy. It is not yet entirely clear how this complex interplay is regulated and which molecular pathways are responsible for each particular outcome. This relationship is particularly important in cancer cells, which are often exposed to nutrient imbalances in the tumour microenvironment and use both autophagy and LDs for protection against stress. The aim of the present master thesis was to investigate some basic connections between autophagy/lipophagy and LDs in highly invasive breast cancer cells exposed to nutrient deficiency. To achieve this goal, we optimized some already established and tested some new methods for visualization and quantification of the interactions between autophagosomal structures and LDs by live-cell confocal microscopy. We found that autophagy is active in breast cancer cells not only during severe starvation in the absence of amino acids and serum, but also during mild starvation in serum-free medium. We show that inhibition of late stages of autophagy with bafilomycin A1 or chloroquine leads to a decrease in LD content during severe starvation, suggesting the possibility that autophagy is involved in LD biogenesis under these conditions. On the other hand, live-cell confocal microscopy experiments revealed that autophagosomal and lysosomal structures colocalize with LDs during mild but not during severe starvation, suggesting that lipophagic breakdown of LDs is active only in the milder, serum-free starvation conditions. Namely, serum-starved, but not amino acid- and serum-starved breast cancer cells, displayed a marked overlap of autophagosomal (mRFP-LC3) and lysosomal (LAMP1-RFP) fluorescent protein markers and LDs. We also found that late-stage autophagy inhibitors do not affect breast cancer cell survival, at least within the relatively short 24-hour starvation period tested here. In summary, our research work points to a dual, starvation conditions-dependent role of autophagy in the biogenesis and breakdown of LDs in breast cancer cells and offers new clues that will contribute to the explanation of the dynamics of cancer cell stress responses. |
