Thomas Williams

• Doctor of Philosophy
• PostDoc Position at University of Oxford

When cells are stressed, bulk translation is often downregulated to reduce energy demands while stress-response proteins are simultaneously upregulated. To promote proteasome assembly and activity and maintain cell viability upon TORC1 inhibition, 19S regulatory-particle assembly chaperones (RPACs) are selectively translated. However, the molecular...

Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum In Dictyostelium, macropinocytic cups are organized by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3...

Macropinocytosis is a conserved endocytic process used by Dictyostelium amoebae for feeding on liquid medium. To further Dictyostelium as a model for macropinocytosis, we developed a high-throughput flow cytometry assay to measure macropinocytosis, and used it to identify inhibitors and investigate the physiological regulation of macropinocytosis....

ELife digest Cells can use a process known as macropinocytosis to take up fluid from their surroundings. This process plays an important role in many situations. For example, it allows human immune cells to sample their environment to search for harmful microbes and viruses and helps cancer cells to collect more nutrients so that they can grow more...

Macropinocytosis—the large-scale, non-specific uptake of fluid by cells—is used by Dictyostelium discoideum amoebae to obtain nutrients. These cells form circular ruffles around regions of membrane defined by a patch of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the activated forms of the small G-proteins Ras and Rac. When this ruffle cl...

When stressed, cells need to adapt their proteome to maintain protein homeostasis. This requires increased proteasome assembly. Increased proteasome assembly is dependent on increased production of proteasome assembly chaperones. In S. cerevisiae, inhibition of the growth-promoting kinase complex TORC1 causes increased proteasome assembly chaperone...

How well do you know the parts of the cell? Can you match them quickly? Play along with this dobble-inspired game to really get to grips with cells, and the bits that make them! Play with 2-4 people age 2+, with each game lasting around 5 minutes.

Cell stresses occur in a wide variety of settings: in disease, during industrial processes, and as part of normal day‐to‐day rhythms. Adaptation to these stresses requires cells to alter their proteome. Cells modify the proteins they synthesize to aid proteome adaptation. Changes in both mRNA transcription and translation contribute to altered prot...

Did you know lipids (fats) are an essential part of life? Explore how the cells in your body store lipids when there are too many of them by building your own lipid droplets the same way cells do! A game for one or more players aged 4+, at least one of whom should be able to read! Each game lasts around 10 minutes.

When stressed, cells need to adapt their proteome to maintain protein homeostasis. This requires increased proteasome assembly. Increased proteasome assembly is dependent on increased proteasome assembly chaperone expression. In S. cerevisiae , inhibition of the growth-promoting kinase complex TORC1 causes increased proteasome assembly chaperone ex...

Proteome adaptation is key to cells surviving stresses. Increased translation of proteasome assembly chaperones (PACs) is critical for increasing proteasome assembly and cell degradative capacity. The endocytic protein Ede1 recruits PAC mRNA to cortical actin patches in Saccharomyces cerevisiae for translation upon stress. We show, through genetic...

Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated by tight control of TORC1 activity. Upon TORC1 inhibition, protein degradation is increased and protein synthesis is reduced through inhibition of translation initiation to maintain cell viability. Here, we show that the ribosome‐associated complex (RAC)/Ssb cha...

The homohexameric p97 complex, composed of Cdc48 subunits in yeast, is a crucial component of protein quality control pathways including ER-associated degradation. The complex acts to segregate protein complexes in an ATP-dependent manner, requiring the engagement of cofactor proteins that determine substrate specificity. The function of different...

All living things from whole people to single cells and even viruses have life cycles. Explore the weird and wonderful world of life cycles at the level of the cell in this top trumps inspired game. Print and cut out the cards, then play anywhere you want!

Roll the dice to build your cell! To survive dangers, your cell needs sensors, messengers, an organiser, responders, and recyclers. The fastest person to build up the cell wins! A fun cell biology game, based on the classic beetle drive.

Eukaryotic cells achieve proteostasis by ensuring their protein requirements are met through tight control of TORC1 activity. Upon TORC1 inhibition, degradative activity is increased, and protein synthesis is reduced through inhibition of translation initiation, to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ss...

Cell Survival is a free print and play family Cell Biology game for 3+ years. Players compete to keep their cell alive using the cells defences to defeat cell dangers. Comes with activity sheets, a story book, and several game modes to keep things interesting! Download for free at https://doi.org/10.20933/100001271

Cell Survival is a free print and play curriculum-linked Cell Biology game for 8-13 year olds. Players compete to keep their cell alive using the cells defences to defeat cell dangers. Comes with lesson plan, supporting videos, activity sheets, a story book, and several game modes to keep things interesting! Download for free at https://doi.org/10....

Cell homeostasis is maintained in all organisms by the constant adjustment of cell constituents and organisation to account for environmental context. Fine-tuning of the optimal balance of proteins for the conditions, or protein homeostasis, is critical to maintaining cell homeostasis. Actin, a major constituent of the cytoskeleton, forms many diff...

The conserved protein kinase complex TORC1 is a central regulator of protein homeostasis. TORC1 is active under nutrient replete, unstressed conditions and promotes bulk protein synthesis, cell growth, and proliferation. Many stresses inactivate TORC1, including nutrient starvation. When TORC1 is inactivated, bulk protein synthesis is inhibited, an...

Cells require thousands of unique proteins to be in the right place, at the right time, in the right amounts and with the right modifications. They do this through several processes collectively known as protein homeostasis. TORC1 is a principal regulator of protein homeostasis, coordinating protein synthesis and degradation. The proteasome, compos...

When cells are stressed, bulk translation is often downregulated to reduce energy demands whilst stress-response proteins are simultaneously upregulated. 19S Regulatory Particle Assembly-Chaperones (RPACs) are selectively translated upon TORC1 inhibition to promote proteasome assembly and activity, maintaining cell viability. However, the molecular...

Signaling from chemoattractant receptors activates the cytoskeleton of crawling cells for chemotaxis. We show using phosphoproteomics that different chemoattractants cause phosphorylation of the same core set of around 80 proteins in Dictyostelium cells. Strikingly, the majority of these are phosphorylated at an [S/T]PR motif by the atypical MAP ki...

Table S4. GO Enrichment and Core Phosphoproteome Data, Related to Figures 1 and 2 Excel spreadsheet containing full results of GO enrichment analysis for cAMP and folate SILAC experiments, class I phosphorylation sites identified in both cAMP and folate experiments, and class I phosphorylation sites matching the p[S/T]PR motif.

Table S5. Phosphoproteomics with erkB- Cells, ErkA In Vitro Phosphorylation Data, Related to Figures 4 and S4 Excel spreadsheet containing phosphoproteomic MS data for erkB- experiment and MS data for ErkA in vitro phosphorylation experiment.

Excel spreadsheet containing details of oligonucleotides used in this study.

Video of F-actin structures in Ax2 wild-type and erkB− undifferentiated amoebae during random motility. Maximum intensity projection of a confocal z-stack. Scale bar, 10 μm. Cells were imaged at 2 frames per min for 15 min. Movie frame rate 15 fps.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers an...

Macropinocytosis is used by a variety of amoebae for feeding on liquid medium. The amoebae project cups and ruffles from their plasma membrane, driven by actin polymerization, and eventually fuse these back to the membrane, entrapping droplets of medium into internal vesicles. These vesicles are of up to several microns in diameter and are processe...

Macropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium , macropinocytic cups are organised by ‘macropinocytic patches’ in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and di...

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, ove...

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild t...

Expression of fluorescent proteins from an extra-chromosomal vector in non-axenic wild-type cells, including new brighter fluorescent proteins suitable for difficult samples. (A) Quantification of mCherry fluorescence intensity after transfection of the extrachromosomal plasmid pDM1208 in DdB cells. Cells were selected with different concentrations...

Efficient inducible expression in bacterially cultured cells. Adaptation of the doxycycline inducible expression system to cells grown on bacteria. (A-B) Dose-response curves for GFP (pDM1047) and mCherry (pDM1046) expression induced by doxycycline. NC4 cells were transfected with the respective plasmids and cultured in the absence of doxycycline (...

The protocols for bacterially grown cells are also efficient under axenic conditions. (A) Efficiencies of knock-out generation, knock-in to the act5 safe locus and knock-in to targeted loci. The number of correct clones is plotted against the total number of clones obtained. Knock-outs are displayed in blue, act5 knock-ins in white and targeted kno...

Transformations in HL5 medium using the new established electroporation conditions. (DOCX)

Cultivation of Dictyostelium discoideum cells in bacterial suspension (OD = 2). (MOV)

Southern Blot act5::mScarlet KI clones. (TIF)

Validation of knock-ins at the act5 locus. Homogenous expression through single copy integration. (A) Scheme for the integration of the act5-KI construct of mScarlet (pPI419) into the act5 gene locus. The recombination arms are displayed in orange, mScarlet in red, the act5 gene and the Hygromycin resistance cassette in white. In black the upstream...

Comparison of the fluorescence intensity of GFP expressed as an act5 knock-in before and after removal of the resistance cassette, and from an extra-chromosomal expression vector. (A) Flow cytometry analysis of cellular fluorescence of four independent act5::GFP knock-in clones obtained by transfection of pDM1513 in AX2 cells. (B) Quantification of...

Alternative knock-in system allowing step-wise assembly. (A) Scheme of an alternative knock-in system. The two recombination-arms are shown in blue. The 5’ arm is cloned using the BglII/SpeI sites while the 3’ arm is added using SalI/SacI. The SpeI site directly follows the desired tag (light green). The cloned knock-in is terminated by an act8-ter...

DNA sequences of the plasmids. (PDF)

Southern Blot rasS- clones. (TIF)

Preparation of food bacteria. (PDF)

Extrachromosomal expression. (PDF)

Knock-in at the act5 locus in different Dictyostelium strains reproducibly yields high expression with minimal cell-to-cell variability. (A) Images of four independent act5::mCherry knock-ins. Clones were generated in the NC4 background using pDM1514 and images taken by confocal microscopy. An overlay of the red fluorescence channel and the DIC is...

Organelle markers: act5 knock-in of histone H2B as a nuclear marker. (A) Flow cytometry analysis of act5::H2B-mCherry KI clones, generated in an AX2 background. (B) Quantification of the experiment shown in (A), replicated 3 times. (C) Comparison of cell-to-cell fluorescence of knock-in clones, measured by confocal microscopy (50 cells each). (D) F...

Knock-in to the HSPC300 component of the SCAR/WAVE complex. (A) Confocal micrographs of randomly moving cells of HSPC300-GFP knock-in clones grown in bacterial suspension. Four independent clones show similar patterns and near-equal fluorescence intensities. Scale bar 10 μm. (B) Fluorescence intensity of individual cells of the four clones was dete...

Cell lines used in the paper. (DOCX)

Other plasmids used in the paper. (DOCX)

Oligonucleotides used in this paper. (DOCX)

Dictyostelium discoideum feeding on Klebsiella aerogenes bacteria. (AVI)

act5::H2B-mCherry expressing AX2 cells in SorMC buffer. (AVI)

In a role distinct from and perhaps more ancient than that in signal transduction, PIP3 and Ras help to spatially organize the actin cytoskeleton into macropinocytic cups. These large endocytic structures are extended by actin polymerization from the cell surface and have at their core an intense patch of active Ras and PIP3, around which actin pol...

Macropinocytosis is a conserved endocytic process used by Dictyostelium amoebae for feeding on liquid medium. To further Dictyostelium as a model for macropinocytosis, we developed a high-throughput flow cytometry assay for macropinocytosis, and used it to identify inhibitors and investigate the physiological regulation of macropinocytosis. Dictyos...

Macropinocytosis is a means by which cells non-specifically take up medium into cytoplasmic vesicles greater than 0.2 μm in diameter. Actin is polymerised around a patch of active Ras, Rac and PIP3 to form a cup that closes, internalising a droplet of extracellular fluid. Macropinocytosis is clinically important for immune defence, pathogen invasio...