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Daniel L Purich

Publications and source records attributed to Daniel L Purich.

13 recordsLinked to original sources
PubMed2006

Nematode sperm motility: nonpolar filament polymerization mediated by end-tracking motors.

In nematode sperm cell motility, major sperm protein (MSP) filament assembly results in dynamic membrane protrusions in a manner that closely resembles actin-based motility in other eukaryotic cells. Paradoxically, whereas actin-based motility is driven by addition of ATP-bound actin subunits onto actin filament plus-ends located at the cell membrane, MSP dimers assemble from solution into nonpolar filaments that lack a nucleotide binding site. Thus, filament polarity and on-filament ATP hydrolysis, although essential for actin-based motility, appear to be unnecessary for membrane protrusions by MSP. As a potential resolution to this paradox, we propose a model for MSP filament assembly and force generation by MSP filament end-tracking proteins. In this model, ATP hydrolysis drives affinity-modulated, processive interactions between membrane-associated proteins and elongating filament ends. However, in contrast to the "actoclampin" model for actin filament end-tracking motors, ATP activates the tracking protein (or a soluble cofactor) rather than the MSP subunits themselves (in contrast to activation of actin subunits by ATP binding). The MSP end-tracking model predicts properties that are consistent with several key observations of MSP-based motility, including persistent membrane attachment, polymerization of filament ends at the membrane with depolymerization of free-filament ends away from the membrane, as well as a saturating dependence of polymerization rate on the concentration of non-MSP soluble cytoplasmic components.

Actins↗
PubMed2006

Diffusion rate limitations in actin-based propulsion of hard and deformable particles.

The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.

Actins↗
PubMed2005

Listeria's right-handed helical rocket-tail trajectories: mechanistic implications for force generation in actin-based motility.

Listeria monocytogenes forms right-handed helical rocket tail trajectories during actin-based motility in cell-free extracts, and this stereochemical feature is consistent with actoclampin's affinity-modulated, clamped-filament elongation model [Dickinson and Purich, 2002: Biophys J 82:605-617]. In that mechanism, right-handed torque is generated by an end-tracking molecular motor, each comprised of a filament barbed end and clamping protein that processively traces the right-handed helix of its filament partner. By contrast, torque is not a predicted property of those models (e.g., elastic propulsion, elastic Brownian ratchet, tethered ratchet, and insertional polymerization models) requiring filament barbed ends to depart/detach from the motile object's surface during/after each monomer-addition step. Helical trajectories also explain why Listeria undergoes longitudinal-axis rotation on a length-scale matching the helical periodicity of Listeria's rocket tails.

Actins↗
PubMed2005

Structural insights into Alzheimer filament assembly pathways based on site-directed mutagenesis and S-glutathionylation of three-repeat neuronal Tau protein.

Although Tau and MAP2 readily assemble into straight filaments (SFs), Tau's unique ability to form paired-helical filaments (PHFs) may offer clues as to why Tau's microtubule-binding region (MTBR) is the exclusive building block of the neurofibrillary tangles that accumulate during Alzheimer's disease. To learn more about the factors permitting Tau to form both SFs and PHFs, we investigated the microtubule binding, thiol oxidation, and polymerization reactions of the monomer and dimer forms of Tau and MAP2 MTBRs. This review focuses on electron microscopic evidence (1) that facilitated the identification of amino acid residues within 3-repeat Tau that promote PHF formation; and (2) provided experimental evidence for the polymerization of S-glutathionylated three-repeat Tau, a reaction that unambiguously demonstrates that disulfide-linked Tau-S-S-Tau dimer formation is not a compulsory step in filament assembly. We also consider these findings within the context of current views on the genetic and biochemical basis of Tau fibrillogenesis.

Alzheimer Disease↗
PubMed2004

Force generation by cytoskeletal filament end-tracking proteins.

Force generation in several types of cell motility is driven by rapidly elongating cytoskeletal filaments that are persistently tethered at their polymerizing ends to propelled objects. These properties are not easily explained by force-generation models that require free (i.e., untethered) filament ends to fluctuate away from the surface for addition of new monomers. In contrast, filament end-tracking proteins that processively advance on filament ends can facilitate rapid elongation and substantial force generation by persistently tethered filaments. Such processive end-tracking proteins, termed here filament end-tracking motors, maintain possession of filament ends and, like other biomolecular motors, advance by means of 5'-nucleoside triphosphate (NTP) hydrolysis-driven affinity-modulated interactions. On-filament NTP hydrolysis/phosphate release yields substantially more energy than that required for driving steady-state assembly/disassembly of free filament ends (i.e., filament treadmilling), as revealed by an energy inventory on the treadmilling cycle. The kinetic and thermodynamic properties of two simple end-tracking mechanisms (an end-tracking stepping motor and a direct-transfer end-tracking motor) are analyzed to illustrate the advantages of an end-tracking motor over free filament-end elongation, and over passive end-trackers that operate without the benefit of NTP hydrolysis, in terms of generating force, facilitating rapid monomer addition, and maintaining tight possession of the filament ends. We describe an additional cofactor-assisted end-tracking motor to account for suggested roles of cofactors in the affinity-modulated interactions, such as profilin in actin-filament end-tracking motors and EB1 in microtubule end-tracking motors.

Actins↗
PubMed2003

Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc.

Although motile endocytic vesicles form actin-rich rocket tails [Merrifield et al., 1999: Nature Cell Biol 1:72-74], the mechanism of intracellular organelle locomotion remains poorly understood. We now demonstrate that bone marrow macrophages treated with lanthanum and zinc ions, well-known secretagogue antagonists, reliably exhibit vesicle motility. This treatment results in accentuated membrane ruffling and the formation of phagosomes and early endosomes that move rapidly through the cytoplasm by assembling actin filament rocket tails. Protein-specific immunolocalization demonstrated the presence of Arp2/3 complex in the polymerization zone and throughout the actin-rich tail, whereas N-WASP was most abundant in the polymerization zone. Although Arp2/3 and N-WASP play essential roles in nucleating filament assembly, other processes (i.e., elongation and filament cross-linking) are required to produce forces needed for motility. Efficient elongation was found to require zyxin, VASP, and profilin, proteins that interact by means of their ABM-1 and ABM-2 proline-rich motifs. The functional significance of these motifs was demonstrated by inhibition of vesicle motility by the motif-specific ABM-1 and ABM-2 analogues. Furthermore, lanthanum/zinc treatment also facilitated the early onset of actin-based vaccinia motility, a process that also utilizes Arp2/3 and N-WASP for nucleation and the zyxin-VASP-profilin complex for efficient elongation. Although earlier studies using cell extracts clouded the role of oligoproline sequences in activating the polymerization zone, our studies emphasize the importance of evaluating motility in living cells.

Actin-Related Protein 2↗
PubMed2002

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.

Actins↗
PubMed2002

In vitro assembly of Alzheimer-like filaments. How a small cluster of charged residues in Tau and MAP2 controls filament morphology.

Although the microtubule-binding regions (MTBRs) of both Tau and MAP2 can undergo self-assembly into straight filaments (SFs) in vitro, only the Tau MTBR forms paired helical filaments (PHFs). Moreover, Tau appears to be the exclusive building block of the neuropathic filaments observed in Alzheimer's disease and certain frontotemporal dementias (FTDs). Despite significant conservation in the MTBR sequences, there are two persistently different stretches of amino acids (designated here as Module-A and Module-B) between Tau and MAP2 from a number of organisms. To evaluate the role of charged residues in these modules as potential morphology-specifying elements, we used site-directed mutagenesis to replace selected residues within the MAP2 MTBR by residues at corresponding positions in Tau. We then employed electron microscopy to determine the frequency of occurrence of SF and PHF morphology in filaments assembled from these mutant microtubule-binding regions. Our experimental results indicate that a very small number of residues are especially significant determinants of filament morphology; this inference is also supported by the observation that site-directed substitutions of individual Tau residues into MAP2 Module-B likewise result in the formation of PHF-like structures. Because the Module-B in Tau contains two naturally occurring FTD mutations, residues in this region may play a critical role in neuropathic filament assembly.

Alzheimer Disease↗
PubMed2002

Actin-based phagosome motility.

Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella. By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility. Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility. Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads. Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes [Merrifield et al., 1999: Nat. Cell Biol. 1:72-74.]. Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation.

Actins↗
PubMed2002

Clamped-filament elongation model for actin-based motors.

Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility.

Actins↗