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| Status |
Public on Nov 18, 2023 |
| Title |
Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specifics aspects driving this conservation are unclear. Here, we screened a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants revealed distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explained sequence constraints on phosphoregulation, which were instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We found remarkably loose sequence requirements for actin binding and phosphoinhibition, but collectively they restricted the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.
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| Overall design |
The S. cerevisiae TeTO7-COF1 strain31 (Horizon Discovery TH_5610) was transformed sequentially with pRS415-GAL1-FLAG-LIMK1 and the cofilin library plasmid pool and frozen in aliquots to conduct replicate screens. For each screen, transformed yeast were pooled and grown at 30 °C with shaking in liquid culture with selective media (SC-His- Leu) containing 2% glucose at a starting OD600 of 0.1. After 4 - 5 population doublings, a portion was diluted to an OD600 of 0.1 with SC-His-Leu media containing 2% raffinose and 10 mg/L DOX. After the culture reached an OD600 of 1 - 1.5, a portion was reserved for plasmid recovery (T0 sample), and the remaining culture was split and diluted into SC-His-Leu liquid media containing 10 mg/L DOX and either 2% glucose or 2% raffinose/1% galactose to OD600 = 0.1. Cultures were subjected to three growth and dilution cycles in which they were grown to an OD600 of 1 - 2 and diluted back to an OD600 of 0.1, saving a portion for plasmid recovery at each step (T1 – T3 samples). After the screen was complete, plasmids were recovered from cell pellets using a QIAGEN Spin Miniprep kit. The N-terminal variable region of the plasmid pool was PCR amplified to attach sequencing adaptors and add barcodes and sequenced on an Illumina NovaSeq instrument. The relative representation of each cofilin variant at each timepoint was calculated by dividing the number of reads that variant by the total read count. The enrichment or depletion of a cofilin-1 library sequence in the absence of LIMK1 induction was calculated as the ratio of its relative representation at T0 and T2 in glucose. Enrichment or depletion following LIMK1 induction was calculated from timepoints T1 and T3 in glucose or galactose. Sequences were ranked by the average log fold changes in representation across three replicate screens. Data were processed using Microsoft Excel.
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| Web link |
https://www.nature.com/articles/s41467-024-45878-9
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| Contributor(s) |
Sexton JA, Turk BE |
| Citation(s) |
38365893 |
| Submission date |
Sep 05, 2023 |
| Last update date |
Feb 17, 2024 |
| Contact name |
Ben Turk |
| E-mail(s) |
ben.turk@yale.edu
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| Organization name |
Yale University
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| Department |
Pharmacology Department
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| Lab |
Ben Turk Lab
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| Street address |
333 Cedar Street, SHM B386
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06520 |
| Country |
USA |
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| Platforms (1) |
| GPL27812 |
Illumina NovaSeq 6000 (Saccharomyces cerevisiae) |
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| Samples (14)
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| GSM7763152 |
Starting Cofilin Library |
| GSM7763153 |
LIMK1 Cofilin Library Screen Replicate 1 Timepoint 0 Glucose |
| GSM7763154 |
LIMK1 Cofilin Library Screen Replicate 1 Timepoint 0 Raffinose DOX |
| GSM7763155 |
LIMK1 Cofilin Library Screen Replicate 1 Timepoint 1 |
| GSM7763156 |
LIMK1 Cofilin Library Screen Replicate 1 Timepoint 2 |
| GSM7763157 |
LIMK1 Cofilin Library Screen Replicate 1 Timepoint 3 |
| GSM7763158 |
LIMK1 Cofilin Library Screen Replicate 2 Timepoint 0 |
| GSM7763159 |
LIMK1 Cofilin Library Screen Replicate 2 Timepoint 1 |
| GSM7763160 |
LIMK1 Cofilin Library Screen Replicate 2 Timepoint 2 |
| GSM7763161 |
LIMK1 Cofilin Library Screen Replicate 2 Timepoint 3 |
| GSM7763162 |
LIMK1 Cofilin Library Screen Replicate 3 Timepoint 0 |
| GSM7763163 |
LIMK1 Cofilin Library Screen Replicate 3 Timepoint 1 |
| GSM7763164 |
LIMK1 Cofilin Library Screen Replicate 3 Timepoint 2 |
| GSM7763165 |
LIMK1 Cofilin Library Screen Replicate 3 Timepoint 3 |
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| Relations |
| BioProject |
PRJNA1013251 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE242403_Primer_Barcode_Index_LIMK1_Cofilin_Screen.xlsx |
15.3 Kb |
(ftp)(http) |
XLSX |
| GSE242403_Raw_Counts_LIMK1_Cofilin_Screen.xlsx |
3.2 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
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