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bioRxiv2024

Pancreatic cancer cells exchange ribosomes through tunneling nanotubes

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/597772v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1f66716org.highwire.dtl.DTLVardef@1ea14eeorg.highwire.dtl.DTLVardef@1044da2org.highwire.dtl.DTLVardef@184d8a3_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG Key pointsO_LIpancreatic tumour cell lines and cells from patient biopsies form tunnelling nanotubes in 2D culture C_LIO_LIformation of tunnelling nanotubes is promoted by gemcitabine C_LIO_LIpolyadenylated mRNAs, ribosomal components and assembled ribosomes are present in tunnelling nanotubes C_LIO_LIribosomes and their components are transferred via nanotubes to acceptor cells C_LIO_LIsilencing of ribosomal proteins S6 and L24 reduces the number of assembled ribosomes C_LIO_LIglobal protein synthesis and number of ribosomes in pancreatic cancer cells with silenced ribosomal proteins increases when co-cultured with translationally unimpaired cells C_LI BackgroundPancreatic ductal adenocarcinoma (PDAC) is considered as one of the deadliest types of cancer. Tunnelling nanotubes (TNTs) are thin, membranous, intercellular communication structures observed in normal and cancer cells, where they mediate the exchange of intracellular material and promote cell fitness, cancer spread and treatment resistance. ResultsPDAC cells increase the formation of TNTs upon exposure to gemcitabine. In the PANC-1 cell line and in tumour explants from patients, we observe polyadenylated mRNA, 5.8S rRNA, ribosomal proteins and assembled 80S ribosomes within the TNTs. Using HaloTag-labelled small ribosomal subunit component RPS9 we demonstrate the transport of ribosomes via TNTs into acceptor cells. Downregulation of ribosomal proteins S6 and L24 decreases the number of assembled ribosomes and the global protein translation in PDAC cells, while a co-culture with translationally unimpaired cells partially restores protein synthesis in cells with impaired protein translation. ConclusionsPDAC cells can exchange components of the protein translation machinery and mRNA. The intercellular transfer of these components causes a partial restoration of protein translation in cells with impaired protein synthesis, which may contribute to the resilience of pancreatic cancer cells, highlighting the potential of targeting TNT dynamics as a therapeutic approach for PDAC.

cancer biology↗

bioRxiv2021

Cell-Tak coating may cause mis-normalization of Seahorse metabolic flux data

Metabolic flux investigations of cells and tissue samples are a rapidly advancing tool in diverse research areas. Reliable methods of data normalization are crucial for an adequate interpretation of results and to avoid a misinterpretation of experiments and incorrect conclusions. The most common methods for metabolic flux data normalization are to cell number, DNA and protein. Data normalization may be affected by a variety of factors, such as density, healthy state, adherence efficiency, or proportional seeding of cells. The mussel-derived adhesive Cell-Tak is often used to immobilize poorly adherent cells. Here we demonstrate that this coating may strongly affect the fluorescent detection of DNA leading to an incorrect and highly variable normalization of metabolic flux data. Protein assays are much less affected and cell counting can virtually completely remove the effect of the coating. Cell-Tak coating also affects cell shape in a cell line-specific manner and may change cellular metabolism. Based on these observations we recommend cell counting as a gold standard normalization method for Seahorse metabolic flux measurements with protein content as a reasonable alternative.

biochemistry↗

bioRxiv2021

Selective and reversible disruption of mitochondrial inner membrane protein complexes by lipophilic cations

Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.

biochemistry↗