Initiation of ensemble kinesin-3 motility is regulated by the rigidity of cargo-motor attachment

AbstractIntracellular cargo transport is powered by molecular motors that move on their respective filamentous tracks. A key component in this process is the tether between cargo and motor, which is often connected by long slender coiled-coils. Several studies have identified mechanisms that regulate cargo transport and can be broadly categorized into regulation of the motor ATPase activity by autoinhibition, cargo adapters and modifications in the cytoskeletal tracks. The regulatory effects of cargo-motor linkers have been described in kinesin-3 subfamily motors. However, the effects of cargo-motor linker rigidity on ensemble cargo transport has not been explored. Here we have built a DNA origami scaffold, which can be tethered with multiple kinesin-3 motors using either single or double-stranded DNA linkages, mimicking rigid versus flexible cargo-motor linkages. Using this system, we show that regardless of the motor numbers attached to the cargo, only linkers with a lesser degree of freedom allow motors to engage with microtubule tracks. Together, our work identifies that the rigidity of cargo-motor linkages influences motor motility. This opens up the possibilities to identify new factors that can influence the rigidity of cargo-motor linkages that in turn can regulate intracellular cargo transport.</jats:p

Contents

Structural studies on mammalian septins – New insights into filament formatio

Contribution of cation-π interactions to protein stability

Calculations predict that cation-π interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-p interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-π interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx). In each case, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-p pair was replaced by Leu. Stabilities of wild-type (WT) and mutant proteins were characterized by both thermal and chemical denaturation. Gln and aromatic → Leu mutants were consistently less stable than corresponding Met mutants, reflecting the nonisosteric nature of these substitutions. The strength of the cation-π interaction was assessed by the value of the change in the free energy of unfolding [ΔΔ G° = Δ G°(Met) - Δ G°(WT)]. This ranged from +1.1 to −1.9 kcal/mol (average value −0.4 kcal/mol) at 298 K and +0.7 to −2.6 kcal/mol (average value -1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-π interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than calculated values with an average difference |Δ G°expt - Δ G°calc|av of 2.9 kcal/mol. At room temperature, the data indicate that cation-π interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However, at elevated temperatures, close to typical Tm's, cation-π interactions are generally stabilizing

Tyrosination of α-tubulin controls the initiation of processive dynein-dynactin motility.

Post-translational modifications (PTMs) of α/β-tubulin are believed to regulate interactions with microtubule-binding proteins. A well-characterized PTM involves in the removal and re-ligation of the C-terminal tyrosine on α-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule-binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal α-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells

Tyrosination of α-tubulin controls the initiation of processive dynein-dynactin motility.

Post-translational modifications (PTMs) of α/β-tubulin are believed to regulate interactions with microtubule-binding proteins. A well-characterized PTM involves in the removal and re-ligation of the C-terminal tyrosine on α-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule-binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal α-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells

Structural insights into filament recognition by cellular actin markers

AbstractCellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here we describe the electron cryomicroscopy structures of phalloidin, lifeAct and utrophin bound to F-actin, providing the first high-resolution structures and comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce conformations and lifeAct specifically recognizes ADP-actin state, which can be used as a sensor for distinguishing different nucleotide states of F-actin. The utrophin structural model aided designing minimal utrophin, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin binding proteins. Further offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.</jats:p

Contribution of Cation-\pi Interactions to Protein Stability

Calculations predict that cation-\pi interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-\pi interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-\pi interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx). In each case, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-\pi pair was replaced by Leu.Stabilities of wild-type (WT) and mutant proteins were characterized by both thermal and chemical denaturation. Gln and aromatic \rightarrow Leu mutants were consistently less stable than corresponding Met mutants, reflecting the nonisosteric nature of these substitutions. The strength of the cation-\pi interaction was assessed by the value of the change in the free energy of unfolding [\Delta \Delta G°=\Delta G°(Met)-\Delta G°- (WT)]. This ranged from +1.1 to -1.9 kcal/mol (average value -0.4 kcal/mol) at 298 K and +0.7 to -2.6 kcal/mol (average value -1.1 kcal/mol) at the $T_m$ of each WT. It therefore appears that the strength of cation-\pi interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than calculated values with an average difference $\vert \Delta G^o_{expt}- \Delta G^o_{calc \vert av}$ of 2.9 kcal/mol. At room temperature, the data indicate that cation-\pi interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However, at elevated temperatures, close to typical $T_{m}\hspace{2mm}'s$, cation-\pi interactions are generally stabilizing

Tubulin tyrosine ligase variant perturbs microtubule tyrosination, causing hypertrophy in patient-specific and CRISPR gene-edited iPSC-cardiomyocytes

Hypertrophic cardiomyopathy (HCM) is a hereditary heart condition characterized by either preserved or reduced ejection fraction without any underlying secondary causes. The primary cause of HCM is sarcomeric gene mutations, which account for only 40%–50% of the total cases. Here, we identified a pathogenic missense variant in tubulin tyrosine ligase (TTL p.G219S) in a patient with HCM. We used clinical, genetics, computational, and protein biochemistry approaches, as well as patient-specific and CRISPR gene-edited induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), to demonstrate that the TTL pathogenic variant results in a reduced enzymatic activity and the accumulation of detyrosinated tubulin leading to the disruption of redox signaling, ultimately leading to HCM. Our findings highlight — for the first time to our knowledge — the crucial roles of the TTL variant in cardiac remodeling resulting in disease