Protein sorting and cell surface polarity in yeast

The studies presented here were focused on the understanding of the principles for protein sorting from the Golgi to the cell surface. As a marker protein we used Fus1p, a type I plasma membrane protein that is O-glycosylated on the extracellular domain and plays a role in cell fusion during yeast mating. Additionally, we analyzed mechanisms responsible for asymmetric distribution of Fus1p in mating cells. We demonstrated that the glycans attached to the protein act as a sorting determinant for protein transport to the cell surface. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A similar defect in exocytosis was observed when a Fus1p mutant lacking the O-glycosylated domain was expressed in wild-type cells, however, the cell surface delivery could be rescued if the 33 amino acid portion of the Fus1p ectodomain, containing 15 potentially glycosylated sites was added to the protein. It was previously well documented in epithelial cells that different types of protein glycosylation and association with lipid rafts play a role of determinants for protein delivery to the apical plasma membrane. However, otherwise the machinery responsible for cargo sorting to the apical membrane is poorly understood. Our finding that also in yeast, protein glycosylation can function as a sorting determinant provides a new possibility to investigate underlying mechanisms..

Drifting sub-pulses in two newly discovered pulsars

We have detected the rare phenomenon of stable, drifting sub-pulse behaviour in two pulsars discovered in the recent Swinburne intermediate latitude pulsar survey. The pulsars; PSR J1231-47 and PSR J1919+0134, have approximate periods (P) of 1.873 and 1.6039 seconds respectively. Both pulsars have multi-component profiles, and distinct drifting is observed across them. We have identified a single drift mode in both pulsars: the drift rate for PSR J1231-47 being 5.4(1) ms P^{-1} and 5.8(2) ms P^{-1} for PSR 1919+0134. The drifting is linear across the profile with no departure from linearity at the edges within the sensitivity of our observations.Comment: 4 pages, 2 figures. Accepted by Monthly Notices of the Royal Astronomical Societ

Plasma membrane polarization during mating in yeast cells

The yeast mating cell provides a simple paradigm for analyzing mechanisms underlying the generation of surface polarity. Endocytic recycling and slow diffusion on the plasma membrane were shown to facilitate polarized surface distribution of Snc1p (Valdez-Taubas, J., and H.R. Pelham. 2003. Curr. Biol. 13:1636–1640). Here, we found that polarization of Fus1p, a raft-associated type I transmembrane protein involved in cell fusion, does not depend on endocytosis. Instead, Fus1p localization to the tip of the mating projection was determined by its cytosolic domain, which binds to peripheral proteins involved in mating tip polarization. Furthermore, we provide evidence that the lipid bilayer at the mating projection is more condensed than the plasma membrane enclosing the cell body, and that sphingolipids are required for this lipid organization

Instability of Extremal Relativistic Charged Spheres

With the question, ``Can relativistic charged spheres form extremal black holes?" in mind, we investigate the properties of such spheres from a classical point of view. The investigation is carried out numerically by integrating the Oppenheimer-Volkov equation for relativistic charged fluid spheres and finding interior Reissner-Nordstr\"om solutions for these objects. We consider both constant density and adiabatic equations of state, as well as several possible charge distributions, and examine stability by both a normal mode and an energy analysis. In all cases, the stability limit for these spheres lies between the extremal ($Q = M$) limit and the black hole limit ($R = R_+$). That is, we find that charged spheres undergo gravitational collapse before they reach $Q = M$, suggesting that extremal Reissner-Nordtr\"om black holes produced by collapse are ruled out. A general proof of this statement would support a strong form of the cosmic censorship hypothesis, excluding not only stable naked singularities, but stable extremal black holes. The numerical results also indicate that although the interior mass-energy $m(R)$ obeys the usual $m/R < 4/9$ stability limit for the Schwarzschild interior solution, the gravitational mass $M$ does not. Indeed, the stability limit approaches $R_+$ as $Q \to M$. In the Appendix we also argue that Hawking radiation will not lead to an extremal Reissner-Nordstr\"om black hole. All our results are consistent with the third law of black hole dynamics, as currently understood

Histone deacetylase 4 protects from denervation and skeletal muscle atrophy in a murine model of amyotrophic lateral sclerosis

Background: Histone deacetylase 4 (HDAC4) has been proposed as a target for Amyotrophic Lateral Sclerosis (ALS) because it mediates nerve-skeletal muscle interaction and since its expression in skeletal muscle correlates with the severity of the disease. However, our recent studies on the skeletal muscle response upon long-term denervation highlighted the importance of HDAC4 in maintaining muscle integrity. Methods: To fully identify the yet uncharacterized HDAC4 functions in ALS, we genetically deleted HDAC4 in skeletal muscles of a mouse model of ALS. Body weight, skeletal muscle, innervation and spinal cord were analyzed over time by morphological and molecular analyses. Transcriptome analysis was also performed to delineate the signaling modulated by HDAC4 in skeletal muscle of a mouse model of ALS. Findings: HDAC4 deletion in skeletal muscle caused earlier ALS onset, characterized by body weight loss, muscle denervation and atrophy, and compromised muscle performance, although the main catabolic pathways were not activated. Transcriptome analysis identified the gene networks modulated by HDAC4 in ALS, revealing UCP1 as a top regulator that may be implicated in worsening ALS features. Interpretation: HDAC4 plays an important role in preserving innervations and skeletal muscle in ALS, likely by modulating the UCP1 gene network. Our study highlights a possible risk in considering HDAC inhibitors for the treatment of ALS. Fund: This work was supported by FIRB grant (RBFR12BUMH) from Ministry of Education, Universities and Research, by Fondazione Veronesi, by Sapienza research project 2017 (RM11715C78539BD8) and Polish National Science Center grant (UMO-2016/21/B/NZ3/03638)

Nonthermal nature of incipient extremal black holes

We examine particle production from spherical bodies collapsing into extremal Reissner-Nordstr\"om black holes. Kruskal coordinates become ill-defined in the extremal case, but we are able to find a simple generalization of them that is good in this limit. The extension allows us to calculate the late-time worldline of the center of the collapsing star, thus establishing a correspondence with a uniformly accelerated mirror in Minkowski spacetime. The spectrum of created particles associated with such uniform acceleration is nonthermal, indicating that a temperature is not defined. Moreover, the spectrum contains a constant that depends on the history of the collapsing object. At first sight this points to a violation of the no-hair theorems; however, the expectation value of the stress-energy-momentum tensor is zero and its variance vanishes as a power law at late times. Hence, both the no-hair theorems and the cosmic censorship conjecture are preserved. The power-law decay of the variance is in distinction to the exponential fall-off of a nonextremal black hole. Therefore, although the vanishing of the stress tensor's expectation value is consistent with a thermal state at zero temperature, the incipient black hole does not behave as a thermal object at any time and cannot be regarded as the thermodynamic limit of a nonextremal black hole, regardless of the fact that the final product of collapse is quiescent.Comment: 13 pages, 2 epsf figures, RevTeX 3. Minor changes, version published in PR

Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation

The force-dependent interaction between talin and vinculin plays a crucial role in the initiation and growth of focal adhesions. Here we use magnetic tweezers to characterise the mechano-sensitive compact N-terminal region of the talin rod, and show that the three helical bundles R1-R3 in this region unfold in three distinct steps consistent with the domains unfolding independently. Mechanical stretching of talin R1-R3 enhances its binding to vinculin and vinculin binding inhibits talin refolding after force is released. Mutations that stabilize R3 identify it as the initial mechano-sensing domain in talin, unfolding at ~5 pN, suggesting that 5 pN is the force threshold for vinculin binding and adhesion progression

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5? and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules

Torque Decay in the Pulsar (P,P_dot) Diagram. Effects of Crustal Ohmic Dissipation and Alignment

We investigate the evolution of pulsars in the (P,P-dot) diagram. We first present analytical formulae to follow the evolution of a pulsar using simple exponential models for magnetic field decay and alignment. We then compare these evolutionary tracks with detailed model calculations using ohmic decay of crustal neutron star magnetic fields. We find that, after an initial phase with a small braking index, n, pulsars evolve with enhanced torque decay (n >> 3) for about 1 Myr. The long term evolution depends on the impurity parameter of the crust. If impurities are negligible in older isolated pulsars we expect their true age to be approximately equal to their observed characteristic age, tau = P/(2*P-dot). It is not possible from data to constrain model parameters of the neutron star crust.Comment: A&A in press, 10 pages, 9 figures, extended discussion of the braking inde