Biology subjects

Shaffer, J. M.

Publications and source records attributed to Shaffer, J. M..

6 recordsLinked to original sources

bioRxiv2026

An Atomistic Description of Heterotypic Lipid Exchange by Sec14-like Phosphatidylinositol Transfer Proteins

Lipid transfer proteins (LTPs) are core regulators of the membrane dynamics, lipid signaling and intracellular communication networks that connect every organelle in the eukaryotic cell. ATP-independent lipid exchange reactions are a hallmark activity of these proteins. These remarkable reactions are essential for the important biological functions of LTPs but how lipid exchange is executed is not at all understood. Herein, we focus on phosphatidylinositol transfer proteins (PITPs) of the highly conserved and highly expanded Sec14/CRAL-Trio-like protein superfamily that potentiate phosphatidylinositol-4-phosphate (PtdIns4P) signaling in eukaryotic cells. Using an integrated structural approach, we describe in atomistic detail the lipid exchange reaction of Sec14-like PITPs. The molecular concepts we identify not only yield insights into how these PITPs integrate metabolic activity with PtdIns4P signaling in cells but also provide a framework for interpreting the functional mechanisms of other LTPs of the Sec14/CRAL-Trio superfamily.

biophysics↗

bioRxiv2025

Residue-level mapping of crowding effects on protein phase separation

Proteins can undergo liquid-liquid phase separation to form condensates within the crowded cellular environment. In vitro, polymer-based crowding agents are widely used to mimic this effect, but how their chemical environments influence phase separation compared to native cytosol remains unclear. Here, we use NMR to probe the chemical influences on the intrinsically disordered region of RNA polymerase II under different crowding conditions, including polymer-based crowders, protein-based crowders, and reconstituted E. coli cytosol. We find a general trend of enhanced protein self-interactions across all conditions, but also distinct chemical environments that depend on crowder identity, reflecting changes in preferential interactions. Given the widespread use of polymer crowders in phase separation studies, our results establish a strategy to dissect the microscopic role of crowders and lay the foundation for designing more physiologically relevant in vitro crowding models of protein phase separation. More broadly, this framework enables systematic probing of residue-level environmental influences in complex settings including within the cellular milieu.

biophysics↗

bioRxiv2023

Molecular basis of RanGTP-activated nucleosome assembly with Histones H2A-H2B bound to Importin-9

Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9*H2A-H2B complex, this RanGTP*Imp9*H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP*Imp9*H2A-H2B ternary complex to those in the Imp9*H2A-H2B or Imp9*RanGTP binary complexes. Our data are consistent with the Imp9*H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin*RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9. SignificanceImp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP alone is insufficient to release H2A-H2B. The resulting stable RanGTP*Imp9*H2A-H2B complex gains nucleosome assembly activity as H2A-H2B can be deposited onto an assembling nucleosome. We show that H2A-H2B is allosterically stabilized via interactions with both N- and C-terminal portions of Imp9, reinforcing its chaperone-like behavior. RanGTP binding causes H2A-H2B release from the N-terminal portion of Imp9 only. The newly-exposed H2A-H2B surfaces can interact with DNA or H3-H4 in nucleosome assembly. Imp9 thus plays a multi-faceted role in histone import, storage, and deposition regulated by RanGTP, controlling histone supply in the nucleus and to chromatin.

biophysics↗

bioRxiv2022

Mechanism of RanGTP priming the release of H2A-H2B from Kap114 and Importin-9

Previously we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9*H2A-H2B complex and formation of the ternary RanGTP*Importin-9*H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for nucleosome assembly. Here we show cryo-EM structures of Importin-9*RanGTP and of its yeast homolog Kap114, including Kap114*RanGTP, Kap114*H2A-H2B, and RanGTP*Kap114*H2A-H2B to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9*RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9*H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP*Kap114*H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome. Significance StatementHistones and their chaperone networks are typically conserved in eukaryotes. The yeast importin Kap114 and its human homolog Importin-9 share low sequence identity, but both are primary nuclear import receptors for the core histone heterodimer H2A-H2B. Cryo-EM structures of Kap114*H2A-H2B, Kap114*RanGTP and Importin-9*RanGTP complexes show homologous structure and function for Kap114 and Importin-9. In the nucleus, RanGTP binding to Kap114/Imp9*H2A-H2B does not release H2A-H2B, but RanGTP binds to form an atypical ternary complex. Structure of the ternary RanGTP*Kap114*H2A-H2B complex explains how the GTPase and cargo bind simultaneously to Kap114 and how the presence of Ran in the complex primes H2A-H2B transfer to assembling nucleosomes.

biochemistry↗

bioRxiv2021

A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

Conditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established "lox-stop-lox" approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5' untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied. Summary statementThe Floxed exon (Flexon), a stop cassette that can be inserted flexibly into the coding region of any gene, facilitates Cre-mediated conditional gene expression.

genetics↗

bioRxiv2019

Systematic assessment of commercially available low-input miRNA library preparation kits

High-throughput sequencing is increasingly favoured to assay the presence and abundance of micro RNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. However, although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human biological total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers sequence (UMI) tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of expected miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

bioinformatics↗