Daniel L. Purich

N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.

The Wiskott–Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

Profilin Promotes Barbed-end Actin Filament Assembly without Lowering the Critical Concentration

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin·4.1·Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-β4, both actin and Profilin·Actin (P·A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant (∼10 × 106 m −1 s−1) for actin monomer or Profilin·Actin. When measured in the absence of profilin, actin assembly curves over a 0.7–4 μmthymosin-β4 concentration range fit a simple monomer sequestering model (1 μm K D for Thymosin-β4·Actin). The corresponding constant for thymosin-β4·pyrenyl-Actin, however, was significantly higher (∼9–10 μm), suggesting that the fluorophore markedly weakens binding to thymosin-β4. With solutions of actin (2 μm) and thymosin-β4 (2 or 4 μm), the barbed-end assembly rate rose with increasing profilin concentration (0.7–2 μm). Actin assembly in presence of thymosin-β4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993)Cell 75, 1007–1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-β4·Actin and ushers actin as a Profilin·Actin complex onto growing barbed filament ends.

Microtubule assembly: a review of progress, principles, and perspectives

Publisher Summary Substantial improvements in electron, light, and fluorescence microscopy, as well as the isolation of discrete protein components of the cytoskeleton, have led the way to a much better appreciation of the structural organization of the cytoplasm. Indeed, the lacelike network of thin filaments, intermediate filaments, and microtubules in nonmuscle cells is as familiar today as the organelles identified after the advent of biological electron microscopy. A major means for characterizing and comprehending the emerging principles of cytoskeletal action has been to isolate and define the properties of various constituents. This chapter considers one such component, the microtubule, from the perspective of the theoretical and experimental efforts to elucidate its assembly and disassembly properties. For this purpose, it concentrates on the in vitro self-assembly of microtubules derived from the neural systems.

EQUIL 93: a tool for experimental and clinical urolithiasis

An extensively updated version of the EQUIL software is described. The former version, designated EQUIL2, is widely used to study urolithiasis and related areas of biomineralization. In this report, we discuss recent enhancements which give EQUIL93 an expanded scope of application. This program has been frequently used in studies of the physicochemical processes underlying stone salt crystallization, especially crystal growth and nucleation, but it has also been employed as an aid for in vivo research and as an evaluator of therapeutic measures. We illustrate several new applications, including some outside the urologic realm, and we discuss how the enhanced software can be helpful in stone risk assessments.

Neurotubule assembly at substoichiometric nucleotide levels using a GTP regenerating system

Abstract Tubulin derived from cold depolymerized bovine microtubules has been gel filtered to obtain a tubulin preparation with only 3% of the tubulin dimers containing exchangeable [ 3 H]-guanine nucleotide. In the presence of acetyl-P and bacterial acetate kinase, this preparation polymerizes to form microtubules which are morphologically indistinguishable from microtubules formed in the presence of excess GTP. The extent of microtubule formation at substoichiometric nucleotide levels using the GTP regenerating system exceeds the extent of assembly obtained with excess GTP. It is concluded that the exchangeable guanine nucleotide site can be virtually unoccupied in intact neurotubules and this finding indicates that GDP can “catalyze” tubule assembly in the presence of a GTP regenerating system.

Carbon Monoxide and Nitric Oxide Mediate Cytoskeletal Reorganization in Microvascular Cells via Vasodilator-Stimulated Phosphoprotein Phosphorylation Evidence for Blunted Responsiveness in Diabetes

OBJECTIVE— We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) on the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP), a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation. RESEARCH DESIGN AND METHODS— We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies. RESULTS— In control platelets, CO selectively promotes phosphorylation at VASP Ser-157, whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239, with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets, neither agent resulted in VASP phosphorylation. In nondiabetic EPCs, NO and CO increased phosphorylation at Ser-239 and Ser-157, respectively, but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions, both CO and NO induced phosphorylation at Ser-157 and Ser-239; however, this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose, VASP was redistributed to filopodia-like structures following CO or NO exposure; however, redistribution was dramatically attenuated under high glucose conditions. CONCLUSIONS— Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type–specific VASP phosphorylation, and in diabetes, blunted responses to these agents may lead to reduced vascular repair and tissue perfusion.

Osmotic susceptibility and diffusion coefficient of charged bovine serum albumin

We report measurements of the osmotic susceptibility and mutual diffusion coefficient of solutions of charged bovine serum albumin molecules made using laser light scattering. The measurements were made as a function of solution ionic strength for various macromolecular charge states ranging from −4e to −13e. We find that the osmotic susceptibility data may be interpreted quantitatively in terms of the simple Verwey–Overbeek interaction potential, provided that adequate care is taken in computing the radial distribution function. Current theoretical treatments of the dynamics, with hydrodynamic interactions included to first order in the concentration, are found to deviate significantly (≲50%) from the diffusion coefficient data. The deviations occur under all conditions of charge and ionic strength for which the susceptibility has been reduced to less than half the ideal gas value.

Reversal of Streptozotocin-Induced Diabetes in Mice by Cellular Transduction With Recombinant Pancreatic Transcription Factor Pancreatic Duodenal Homeobox-1: A Novel Protein Transduction Domain–Based Therapy

OBJECTIVE—The key pancreatic transcription factor pancreatic duodenal homeobox-1 (Pdx1), known to control development and maintenance of pancreatic β-cells, possesses a protein transduction domain (PTD) that facilitates its entry into cells. We therefore sought to evaluate the capacity of in vivo–administered recombinant Pdx1 (rPdx1) to ameliorate hyperglycemia in mice with streptozotocin-induced diabetes. RESEARCH DESIGN AND METHODS—Cell entry and transcriptional regulatory properties of rPdx1 protein and its PTD-deletion mutant rPdx1Δ protein, as well as a PTD–green fluorescent protein, were evaluated in vitro. After intraperitoneal rPdx1 injection into mice with streptozotocin-induced diabetes, we assessed its action on blood glucose levels, insulin content, intraperitoneal glucose tolerance test (IPGTT), Pdx1 distribution, pancreatic gene expression, islet cell proliferation, and organ histology. RESULTS—Restoration of euglycemia in Pdx1-treated diabetic mice was evident by improved IPGTT and glucose-stimulated insulin release. Insulin, glucagon, and Ki67 immunostaining revealed increased islet cell number and proliferation in pancreata of rPdx1-treated mice. Real-time PCR of pancreas and liver demonstrated upregulation of INS and PDX1 genes and other genes relevant to pancreas regeneration. While the time course of β-cell gene expression and serum/tissue insulin levels indicated that both liver- and pancreas-derived insulin contributed to restoration of normoglycemia, near-total pancreatectomy resulted in hyperglycemia, suggesting that β-cell regeneration played the primary role in rPdx1-induced glucose homeostasis. CONCLUSIONS—rPdx1 treatment of mice with streptozotocin-induced diabetes promotes β-cell regeneration and liver cell reprogramming, leading to restoration of normoglycemia. This novel PTD-based protein therapy offers a promising way to treat patients with diabetes while avoiding potential side effects associated with the use of viral vectors.

A direct-transfer polymerization model explains how the multiple profilin-binding sites in the actoclampin motor promote rapid actin-based motility☆

The high actin-based motility rates observed in nonmuscle cells require the per-second addition of 400-500 monomers to the barbed ends of growing actin filaments. The chief polymerization-competent species is profilin.actin.ATP (present at 5-40 microM intracellular concentrations), whereas G-actin.ATP is much less abundant ( approximately 0.1-1 microM). While earlier studies unambiguously demonstrated that profilin.actin is highly concentrated within the polymerization zone, profilin-actin localization on the motile surface cannot increase the local solution-phase concentration of polymerizable actin. To explain these high rates of actin polymerization, we present and analyze a novel polymerization model in which monomers are directly transferred to growing filament ends in the actoclampin motor. This direct-transfer polymerization mechanism endows the polymerization zone with properties unavailable to bulk-phase actin monomers, and our model also indicates why profilin is the ideal mobile carrier for actin monomers.

Evidence for the formation of a γ-phosphorylated glutamyl residue in the Escherichia coli acetate kinase reaction☆

Abstract Studies of phosphorylated Escherichia coli acetate kinase, which has been reduced with [ 3 H]sodium borohydride and subjected to acid hydrolysis, suggest that [ 3 H] α-amino-δ-hydroxyvaleric acid is formed with a 20–25% overall yield. This finding is compatible with the formation of a γ-phosphorylated glutamyl residue upon incubation of enzyme with acetyl-P.