Microtubules: Sizing Up the GTP Cap

SummaryThe ‘GTP cap’ of the microtubule has long been postulated to exist, but a recent experiment gives us the first quantitative measurements of the cap size in the cell

The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating

Large conductance, voltage- and Ca2+-activated K+ (BKCa) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four α subunits of BKCa may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BKCa gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BKCa activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BKCa channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition

Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity.

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions

The unusual dynamics of parasite actin result from isodesmic polymerization

Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here, we re-examine the polymerization properties of actin in Toxoplasma gondii (TgACTI), unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. TgACTI polymerization kinetics lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly, and the size distribution of TgACTI filaments in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum‐Stable Membranes with Low Leakage

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5–30‐fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time‐dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.Hybrid synthetic lipids with dramatically reduced leakage properties incorporate many structural features used by Nature to generate stable membranes. Covalent attachment of cholesterol groups to membrane‐spanning tetraether lipids makes it possible to generate stable liposomes with low permeability, while retaining the possibility to support functional biomolecules and deliver liposome‐encapsulated molecules to living cells.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/1/chem201701378.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/2/chem201701378-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/3/chem201701378_am.pd