Publications by authors named "Shae B Padrick"
Article Synopsis
- Hematologic cancers are prevalent in both adults and children, but many patients still face poor outcomes, highlighting the need for new treatments.
- The Wiskott-Aldrich syndrome protein (WASp) plays a crucial role in actin assembly and is primarily found in blood cells; researchers have created a new small molecule, EG-011, that activates its autoinhibited state.
- Trials show that EG-011 effectively combats various blood cancers like lymphoma and leukemia both in isolated tests and in living models, showcasing its unique mechanism related to actin polymerization.
How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover.
View Article and Find Full Text PDFCyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments.
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- The BORG/Cdc42EP family consists of five proteins that bind to Rho GTPases and are increasingly relevant in understanding cell organization and function.
- Recent studies suggest that BORGs play a role in essential body processes and are linked to diseases, particularly cancers, by affecting the cytoskeleton.
- The relationship between the BORGs and Rho GTPase Cdc42 is complex, varying by cell type and condition, highlighting the need for further research to clarify their mechanisms and implications.
Article Synopsis
- The VanRS two-component system is crucial for how enterococci develop resistance to vancomycin, with VanS activating the transcription factor VanR in response to vancomycin exposure.
- Structural analysis of VanS proteins from vancomycin-resistant enterococci revealed they have a common fold shared with other prokaryotic kinases, but attempts to crystallize their ATP-bound forms were unsuccessful.
- The weak binding affinity of these VanS proteins for ATP suggests a potential regulatory mechanism for controlling the expression of resistance genes under normal cellular conditions.
Article Synopsis
- The coordination of microtubules and actin cytoskeletons is crucial for cellular processes like morphogenesis, division, and migration, with septins playing a key role in this crosstalk.
- Septins, a type of GTP-binding protein, localize to both microtubules and actin filaments, facilitating the formation of hybrid structures that enhance cell growth and stability.
- This study reveals that septins promote actin polymerization in conjunction with microtubules, providing insights into cytoskeletal organization and its implications for cell development.
Production and analysis of a mammalian septin hetero-octamer complex.
Cytoskeleton (Hoboken)
November 2020
Article Synopsis
- Septins are filament-forming proteins present in a variety of eukaryotic organisms, playing crucial roles in processes such as cell division and cytoskeletal organization.
- Researchers successfully produced and characterized a specific recombinant mammalian septin hetero-octamer complex (SEPT2/SEPT6/SEPT7/SEPT3), allowing for the observation of filament assembly through fluorescent protein fusion.
- The study provides insights into the organization of the septin complex, showing that SEPT3 is centrally located while SEPT2 is positioned at the ends of the octamer, laying the groundwork for future exploration of septin mechanisms and functions.
Article Synopsis
- Liquid-liquid phase separation in cells is influenced by multivalent and disordered proteins, helping to form membrane-less organelles.
- The Nephrin intracellular domain (NICD), which is an intrinsically disordered protein, induces this phase separation through complex coacervation, creating dense protein-rich droplets.
- The ability of NICD to drive phase separation relies on its overall amino acid composition, specifically regions with high negative charge density and certain aromatic/hydrophobic residues, indicating that this mechanism may be common among other disordered proteins.
Article Synopsis
- CD437 selectively kills cancer cells without harming normal cells through an unknown mechanism.
- Discovering mutations in POLA1 that lead to resistance against CD437 suggests that POLA1 is crucial for the drug's effectiveness.
- Research shows that CD437 inhibits DNA replication, directly targeting POLA1, which may pave the way for developing CD437 as a cancer treatment.
Article Synopsis
- The understanding of cellular functions relies on the equilibrium dissociation constant (KD), which quantifies how macromolecules bind to each other, with a focus on a new method called microscale thermophoresis (MST).
- MST uses temperature gradients created by an infrared laser to track the movement of dye-labeled molecules, allowing researchers to determine KD by analyzing how these molecules change their migration based on their binding states.
- Simulations highlight the limitations of current instrumentation for measuring KD and stress the importance of binding kinetics in MST experiments; best practices and software for data analysis are also discussed.
Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments.
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- The WAVE regulatory complex (WRC) is a large protein complex that is crucial for regulating actin dynamics in cells by activating the Arp2/3 complex.
- Recent advancements in the methods for producing, purifying, and crystallizing WRC have enhanced our understanding of how this complex is activated and interacts with membranes.
- The laboratory has developed reliable techniques to produce enough high-quality WRC for various biochemical and structural studies, but there are still many unanswered questions about its mechanisms.
Article Synopsis
- VopL is a protein from the bacterium Vibrio parahaemolyticus that helps assemble actin filaments by using its structural components, including a specific domain (VCD) and WH2 motifs.
- The crystal structure reveals how the VCD interacts with actin monomers, positioning them in a way that resembles natural filament structures, allowing for the integration of WH2 motifs without conflict.
- This study highlights a potential nucleation mechanism where VopL organizes actin monomers into filament-like structures, resembling the processes of other known nucleators like the Arp2/3 complex and formin proteins.
Article Synopsis
- During cell movement and the process of endocytosis, WASP proteins attached to membranes help start the formation of actin filaments through the Arp2/3 complex, creating branched arrays of filaments that grow towards the membrane.
- By using advanced imaging techniques, researchers discovered that the interaction timing between Arp2/3 and WASP is essential for the growth of new filaments, with WASP proteins needing to detach from Arp2/3 before new growth begins.
- Their findings indicate that while WASP aids in preparing for filament growth, actual growth happens only after WASP is released, allowing for continuous network expansion without interference.
Article Synopsis
- Actin polymerization is key for cellular force generation, controlled by the cell to regulate nucleation and elongation processes.
- Cells can recreate these processes in controlled lab settings (in vitro), which helps study their signaling pathways for actin formation.
- A method using a pyrene fluorophore allows researchers to easily detect and analyze actin filament formation based on increased fluorescence.
Article Synopsis
- Actin filament dynamics in cells are primarily controlled by proteins that initiate filament formation, known as actin nucleation factors.
- The Arp2/3 complex is a key nucleation factor found across eukaryotes and is important for actin filament branching.
- The text outlines a protocol for isolating the Arp2/3 complex from the budding yeast Saccharomyces cerevisiae, resulting in high-purity material for biochemical research.
Article Synopsis
- The Arp2/3 complex is crucial for cell movement and transporting vesicles, functioning as an actin filament nucleator.
- It is utilized in biochemical tests and structural studies due to its importance in cell biology.
- The purification method described involves extracting the complex from bovine thymus tissue as it is a eukaryotic assembly made of seven polypeptides.
Article Synopsis
- GMF promotes the debranching of actin filament networks crucial for processes like cell movement and transport, acting similarly to the cofilin protein.
- The study identifies that GMF interacts with the Arp2/3 complex and uses two distinct binding surfaces to facilitate this debranching process.
- The mechanism of GMF's activity is conserved across different species, indicating its fundamental role in actin dynamics.
Article Synopsis
- Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV) is a technique that allows researchers to analyze protein complexes in mixtures to determine their number, stoichiometry, and shapes.
- MSSV works by simultaneously measuring multiple signals from macromolecular mixtures, transforming data to identify different sedimentation coefficients even in complex reactions with varying lifetimes.
- A new method introduced in this study improves the spectral deconvolution of poorly resolved components using mass conservation principles, making MSSV more versatile for analyzing reversible macromolecular complexes.
Article Synopsis
- The Arp2/3 complex is crucial for nucleating new actin filaments in eukaryotic cells, activated by signals from the WASP protein family.
- Activation involves two separate VCA domains from WASP proteins binding to distinct sites on the Arp2/3 complex, with each delivering an actin monomer.
- This new understanding resolves previous inconsistencies in the literature regarding VCA activation and proposes a revised model for the mechanism of Arp2/3 complex activation.
Article Synopsis
- Understanding the molecular composition of macromolecular complexes requires knowledge of their components' ratios, which can be analyzed using sedimentation velocity (SV) data from analytical ultracentrifuges.
- This study focuses on a new method that utilizes multiple signals (like Rayleigh interferometry and UV spectrophotometry) to distinguish different components in sedimenting complexes and determine their molar ratios.
- The research also presents techniques for designing experiments, evaluating success in spectral discrimination, and highlights practical applications of the method, including deriving equilibrium association constants.
Article Synopsis
- Members of the WASP family influence cell structure by aiding in the formation of actin filaments through the Arp2/3 complex, particularly involving the WAVE protein and its regulatory complex.
- The WAVE regulatory complex (WRC) is inactive until stimulated by Rac GTPase, certain kinases, and phosphatidylinositols, which interact with the complex.
- The study reveals the crystal structure of WRC, showing how intramolecular and intermolecular interactions keep the active site of WAVE inactive and how external signals can promote activity by destabilizing these interactions.
Determination of the stoichiometry of macromolecular assemblies is fundamental to an understanding of how they function. Many different biophysical methodologies may be used to determine stoichiometry. In the past, both sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation have been employed to determine component stoichiometries.
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- WASP family proteins are crucial activators of the Arp2/3 complex, which is essential for actin filament formation.
- They have a specific VCA domain that triggers this activity in response to various signals, including GTPases and lipids.
- The regulation of WASP proteins involves autoinhibition and dimerization, allowing for enhanced activation of the Arp2/3 complex, with findings from WASP and N-WASP likely applicable to the entire WASP family.
Article Synopsis
- The WAVE regulatory complex (WRC) helps transmit signals from Rac GTPase to the Arp2/3 complex, which is involved in actin polymerization.
- We successfully reconstituted WRC from humans and Drosophila, finding it inactive until activated by Rac, which depends on nucleotide presence.
- Our findings clarify how WAVE is inhibited, resolve existing conflicting theories, and highlight shared regulatory mechanisms among proteins in the WAVE/WASP family.