Type-I myosins promote actin polymerization to drive membrane bending in endocytosis

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations. We show that the type-I myosins promote, in a dose-dependent way, the growth and expansion of the actin network, which controls the speed of membrane and coat internalization. We found that this myosin-activity is independent of the actin nucleation promoting activity of myosins, and cannot be compensated for by increasing actin nucleation. Our results suggest a new mechanism for type-I myosins to produce force by promoting actin filament polymerization

Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression

<div><p>Expression of the tissue-type plasminogen activator gene (t-PA; gene name <i>PLAT</i>) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between <i>PLAT</i> methylation and <i>PLAT</i> expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal <i>PLAT</i> gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the <i>PLAT</i> transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the <i>PLAT</i> promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the <i>PLAT</i> promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient <i>PLAT</i> gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.</p></div

Effect of DNA methylation on reporter gene activity from mutated <i>PLAT</i> promoters.

<p>Reporter gene activity was compared after transfection of HT1080 cells with pCpGLprom151 and promoter mutants. MutA: CpGs -121 and -106 mutated; mut B: CpGs -81, -51 and + 94 mutated; mutC: CpGs +27, +42, +50 and +59 mutated. Results are expressed as means ± SEM of four independent experiments.</p

Influence of DNA methylation on transcription factor binding to a double-stranded DNA containing the Sp1 site.

<p>The effect of oligonucleotide methylation on transcription factor binding was assessed using nuclear extracts from HeLa or HT1080 cells and by comparing the effect of different concentrations of unmethylated (C, concentrations given in nM) or methylated competitor oligonucleotides (MC) on the binding of transcription factors to a double-stranded IRD700-labeled unmethylated oligonucleotide GCCAC<b>CG</b>A<u>CCCCACCCCC</u>TGCCTGGA, which includes the Sp1 site at +62 (underlined) immediately preceded by one CpG (in bold). The analysis was done for cells cultured for 24h in culture medium in the absence or presence of 20 nM PMA. The effect of oligonucleotide methylation on transcription factor binding was assessed using nuclear extracts from HeLa cells (A) or HT1080 cells (B) and by comparing the effect of different concentrations of unmethylated (C, concentrations given in nM) or methylated competitor oligonucleotide (MC) on band intensities. Note that the methylated and unmethylated oligonucleotides had comparable efficacies in preventing transcription factor binding to the IRD700 labeled unmethylated Sp1 probe.</p

Methylation of the <i>PLAT</i> promoter and t-PA secretion in HT1080 cell clones.

<p>The methylation state of the promoter region was analyzed for eight HT1080 cell clones differing in their t-PA secretion and in non-cloned HT1080 cells. The figure gives the percentage methylation of the CpGs in the <i>PLAT</i> promoter region (between -647 and + 94, with respect to TSS1). Bar length’s are proportional to the percentage methylation for each position, with the dotted lines indicating 100% methylation. The position of TSS1 and of the promoter interaction sites with CRE, Sp1 and NF1 is indicated at the top of the figure. Secretion of t-PA expressed in ng per million cells over a 24h period by cells cultured in medium alone is given at the left of the figure.</p

Determination of the <i>PLAT</i> gene transcriptional start site (TSS) in different human cell types.

<p>The TSS for the <i>PLAT</i> gene was assessed by 5’RACE analysis in different t-PA producing human cell types: astrocytes, Bowes melanoma cells, HeLa cells, HUVEC and HT1080 cells, both in cells incubated in culture medium alone (—) or in medium containing 20 nM PMA (+). RACE PCR products were analyzed by agarose gel electrophoresis. PCR products were sequenced and two distinct TSS’s were detected: TSS1 corresponding to position 42’207’676 on chromosome 8 in the GRCh38/hg38 assembly (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>) and TSS2, located 110 bp downstream. Note that the intensity of the band corresponding to TSS1 was increased by PMA (20 nM for 24h) in astrocytes and HUVEC. This figure shows representative results for each cell type.</p

Influence of DNA methylation on transcription factor binding to a double-stranded DNA containing the CRE site.

<p>The effect of oligonucleotide methylation on transcription factor binding was assessed using nuclear extracts from HeLa or HT1080 cells and by comparing the effect of different concentrations of unmethylated (C, concentrations given in nM) or methylated competitor oligo-nucleotides (MC) on the binding of transcription factors to the double-stranded IRD700-labeled unmethylated oligonucleotide TTCCTG<b>CG</b>ATTCAA<u>TGACATCA</u><b>CG</b>GCTGTG, which includes the CRE site (underlined) flanked by two CpGs (in bold). Note that the methylated oligonucleotide was capable of reducing band intensities, albeit at a slightly lesser efficacy as the unmethylated oligonucleotide. The dashed arrow indicates the band whose intensity increases with PMA treatment of HeLa cells. The top of the figure shows the EMSA images and the bottom the % inhibition of band intensities when nuclear extracts were co-incubated with the indicated concentrations (in nM) of unmethylated (C) or methylated (MC) unlabeled competitor oligonucleotides.</p

The In Vivo Architecture of the Exocyst Provides Structural Basis for Exocytosis

The structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane

Effect of 5-azacytidine treatment on the degree of methylation of CpG residues in the proximal t-PA promoter profile in the proximal promoter.

<p>Huh7 were cultured for 3 days in the absence (black bars) or presence (white bars) of 2 μM azacytidine. Bar lengths are proportional to the percentage methylation for each position.</p

Effect of trichostatin on nonmethylated and methylated <i>PLAT</i> promoter-driven reporter gene activity.

<p>HT1080 cells were transfected with nonmethylated (NM) and methylated (M) pCpGLprom151 plasmids and reporter gene activity measured in the absence or presence of 3 μM trichostatin (TSA). The empty condition represents reporter gene activity of a pCpGL plasmid lacking a promoter insert. Note that reporter gene activity of methylated pCpGL plasmids was not significantly different from that of the empty plasmids. Results are expressed as means ± SEM of four independent experiments. * p< 0.05</p