The critical importance of spatial and temporal scales in designing and interpreting immune cell migration assays

Intravital microscopy and other direct-imaging techniques have allowed for a characterisation of leukocyte migration that has revolutionised the field of immunology, resulting in an unprecedented understanding of the mechanisms of immune response and adaptive immunity. However, there is an assumption within the field that modern imaging techniques permit imaging parameters where the resulting cell track accurately captures a cell’s motion. This notion is almost entirely untested, and the relationship between what could be observed at a given scale and the underlying cell behaviour is undefined. Insufficient spatial and temporal resolutions within migration assays can result in misrepresentation of important physiologic processes or cause subtle changes in critical cell behaviour to be missed. In this review, we contextualise how scale can affect the perceived migratory behaviour of cells, summarise the limited approaches to mitigate this effect, and establish the need for a widely implemented framework to account for scale and correct observations of cell motion. We then extend the concept of scale to new approaches that seek to bridge the current “black box” between single-cell behaviour and systemic response

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

<div><p>Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.</p></div

Bioinformatics-based prediction of NF-κB sites in eIFH promoters.

<p>Jaspar- and TFSEARCH-based computational analysis predicted putative NF-κB enhancers in the promoter regions of core eukaryotic translation initiation factors.</p

TNF-α induces the recruitment of p65 to eIF4H promoter.

<p>(A) HeLa cells (5×10⁶) were transfected with siRNA control or siRNA p65 (200 pmol). Forty-eight hours post-transfection, cells were 45 min-treated with TNF-α (20 ng/mL), or left untreated. Total RNA was extracted and analysed by qRT-PCR to measure the expression of eIF4H and eIF2S3. Values (mean ± SD, n = 3) are shown. Statistically significant differences between the samples are shown according to Student's <i>t</i>-test (p≤0.01). (B) HeLa cells (3×10⁷) were treated with TNF-α (20 ng/mL) for the indicated time, or left untreated. Chromatin was immunoprecipitated with anti-p65, anti-p50, anti-RelB, anti-c-Rel or IgG, and ChIP eluates were analysed by qRT-PCR.</p

p65-dependent transcriptional activation of eIF4H.

<p>(A) HeLa cells (5×10⁶) were transfected with pRc/CMV-p65 or pRc/CMV-empty vector (5 µg). Forty-eight hours post-transfection, total RNA was extracted and analysed by qRT-PCR to evaluate the expression of the indicated eIF genes. Values (mean ± SD, n = 5) are shown. The asterisk indicates a statistically significant difference between pRc/CMV-p65 and empty vector according to the Student's <i>t</i>-test (<i>p</i>≤0.01). (B) HeLa cells (5×10⁶) were transfected with siRNA control or siRNA p65 (200 pmol). Forty-eight hours post-transfection, total RNA was extracted and analysed by qRT-PCR for the expression of the indicated eIF genes. Values (mean ± SD, n = 5) are shown. The asterisk indicates a statistically significant difference between siRNA p65 and siRNA control according to the Student's <i>t</i>-test (<i>p</i> ≤0.01). (C) Wild type and p65^−/− MEFs (3×10⁵) were lysed, and total RNA was analysed by qRT-PCR for the expression of eIF4H gene. (D) Total cell extracts (20µg) of wild type and p65^−/− MEFs (3×10⁵) were separated by 12% SDS-PAGE and analysed by western blotting using anti-eIF4H, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the wild type taken as 1. (E) Nuclear extracts of wild type and p65^−/− MEFs (5×10⁶ cells) were analysed for the binding activity of the indicated NF-κB subunits to the NF-κB double-stranded oligonucleotide, as measured by ELISA EMSA using the NF-κB Transcription Factor ELISA assay kit (Cayman). (F) Total RNA from tumour cell lines (MDA-MB-231, MCF-7, SH-SY5Y, U251, D54, MC3, DeFew) (3×10⁵ cells) was analysed by qRT-PCR for the expression of eIF4H gene. (G) Whole protein cell extracts (20µg) of the indicated tumour cell lines were separated by 12% SDS–PAGE and analysed by western blotting using anti-eIF4H, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the MDA-MB-231 cells taken as 1. (H) Nuclear extracts of the indicated tumor cell lines (5×10⁶ cells) were analysed for the p65 binding to the NF-κB double-stranded oligonucleotide, using the NF-κB Transcription Factor ELISA assay kit (Cayman).</p

p65 increases the protein synthesis rate in eIF4H-dependent manner

<p>. (A) HeLa cells (3×10⁶) were transfected with pRc/CMV empty vector (5µg), or pRc/CMV-3HA-p65, in presence of siRNA control or siRNA p65 (200 pmol). Twenty-four hours post-transfection, cells were incubated in methionine/cysteine-free medium for 30 min before addition of labelling medium containing [³⁵S]-methionine/cysteine (10 µCi/ml). One hour after protein labelling, the protein synthesis rate was evaluated. Values (mean ± SD, n = 3) are shown. Statistically significant differences between the samples are indicated according to Student's <i>t</i>-test (p≤0.01). (B) Protein extracts (20µg) of transfected HeLa cells shown in (A) were separated by 12% SDS–PAGE, and analysed by western blotting using anti-eIF4H, anti-HA, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the control (Rc/CMV plus siRNA control), taken as 1.</p

p65-dependent modulation of EIF4H protein expression.

<p>(A) HeLa cells (5×10⁶) were transfected with pRc/CMV-3HA-p65, pRc/CMV-3HA-IκB-α, or pRc/CMV empty vector (5µg), and 48h later whole cell extracts were recovered. Protein extracts (20µg) were separated by 12% SDS–PAGE and analysed by western blotting using anti-HA, anti-eIF4H, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the empty vector, taken as 1. (B) HeLa cells (5×10⁶) were transfected with siRNA control, or siRNA p65 (200 pmol), and forty-eight hours post-transfection whole cell extracts were performed. Protein samples (20µg) were separated by 12% SDS–PAGE, and analysed by western blotting using anti-eIF4H, anti-p65, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above siRNA control, taken as 1. (C) HeLa cells (5×10⁶) were 45 min-stimulated with TNF-α (20 ng/mL), or left untreated, washed twice with DMEM, and lysed to perform total extracts and nuclear extracts. Upper panel, total cell extracts (20µg) were separated by 12% SDS–PAGE and analysed by western blotting using anti-eIF4H or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above un-stimulated cells, taken as 1. Lower panel, nuclear extracts were analysed for the p65 binding to the NF-κB double-stranded oligonucleotide by ELISA EMSA.</p