Cooperation between primary cilia signaling and integrin receptor extracellular matrix engagement regulates progenitor proliferation and neuronal differentiation in the developing cerebellum.

Neural progenitors and their neuronal progeny are bathed in extrinsic signals that impact critical decisions like the mode of cell division, how long they should reside in specific neuronal laminae, when to differentiate, and the timing of migratory decisions. Chief among these signals are secreted morphogens and extracellular matrix (ECM) molecules. Among the many cellular organelles and cell surface receptors that sense morphogen and ECM signals, the primary cilia and integrin receptors are some of the most important mediators of extracellular signals. Despite years of dissecting the function of cell-extrinsic sensory pathways in isolation, recent research has begun to show that key pathways work together to help neurons and progenitors interpret diverse inputs in their germinal niches. This mini-review utilizes the developing cerebellar granule neuron lineage as a model that highlights evolving concepts on the crosstalk between primary cilia and integrins in the development of the most abundant neuronal type in the brains of mammals.

Mechanism of Action of Peptides That Cause the pH-Triggered Macromolecular Poration of Lipid Bilayers.

Using synthetic molecular evolution, we previously discovered a family of peptides that cause macromolecular poration in synthetic membranes at low peptide concentration in a way that is triggered by acidic pH. To understand the mechanism of action of these "pHD peptides", here we systematically explored structure-function relationships through measurements of the effect of pH and peptide concentration on membrane binding, peptide structure, and the formation of macromolecular-sized pores in membranes. Both AFM and functional assays demonstrate the peptide-induced appearance of large pores in bilayers. Pore formation has a very steep pH dependence and is also dependent on peptide concentration. In vesicles, 50% leakage of 40 kDa dextrans occurs at 1 bound peptide per 1300 lipids or only 75 peptides per vesicle, an observation that holds true across a wide range of acidic pH values. The major role of pH is to regulate the amount of peptide bound per vesicle. The physical chemistry and sequence of the pHD peptides affect their potency and pH dependence; therefore, the sequence-structure-function relationships described here can be used for the future design and optimization of membrane permeabilizing peptides for specific applications.

High-Resolution AFM-Based Force Spectroscopy.

Atomic force microscopy (AFM)-based force spectroscopy is a powerful technique which has seen significant enhancements in both force and time resolution in recent years. This chapter details two AFM cantilever modification procedures that yield high force precision over different temporal bandwidths. Specifically, it explains a fairly straightforward method to achieve sub-pN force precision and stability at low frequencies (<50 Hz) by removing the metal coatings from a commercially available cantilever. A more involved procedure utilizing a focused ion beam milling machine is required to maintain high force precision at enhanced bandwidths. Both modification methods allow site-specific attachment of biomolecules onto the apex area of the tips for force spectroscopy. The chapter concludes with a comparative demonstration using the two cantilever modification methods to study a lipid-protein interaction.

Conformations and Dynamic Transitions of a Melittin Derivative That Forms Macromolecule-Sized Pores in Lipid Bilayers.

Systematically evolved from the primary active component of bee venom, MelP5 is a lipophilic peptide with important physical properties that differ from wild-type melittin, including the ability to create large equilibrium pores in lipid bilayers at low peptide to lipid ratios. Self-assembly into stable membrane spanning pores makes MelP5 a promising candidate for future applications in the pharmaceutical arena. Despite significant interest, little is known about the mechanism by which MelP5 remodels the lipid bilayer upon binding. We demonstrate by direct atomic force microscope imaging of supported lipid bilayers in solution that MelP5 remodels 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) in one of two ways. It creates either highly localized voids in the bilayer or diffuse nonlocalized thinning. Thinning of the bilayer was measured to be 3.0 ± 1.4 Å (mean ± standard deviation) below the surface of the upper leaflet of the bilayer. Pores, defined as highly localized voids in the bilayer, exhibited several sizes. Approximately 20% of pores exhibited large footprint areas (47 ± 20 nm2) which appear capable of passing bulky macromolecules. The peptide-effected bilayer was observed to reversibly exchange between membrane-thinned and pore states in an apparent dynamic equilibrium. Analysis of time-lapsed images suggested upper and lower bounds (0.2 < τ < 180 s) on the characteristic time scale of transitions between the membrane-thinned and pore states. Moreover, pores were found to colocalize with membrane-thinned regions, a novel observation that is consistent with the notion of cooperativity among membrane-bound peptides when forming pores.

Potent Macromolecule-Sized Poration of Lipid Bilayers by the Macrolittins, A Synthetically Evolved Family of Pore-Forming Peptides.

Pore-forming peptides with novel functions have potential utility in many biotechnological applications. However, the sequence-structure-function relationships of pore forming peptides are not understood well enough to empower rational design. Therefore, in this work, we used synthetic molecular evolution to identify a novel family of peptides that are highly potent and cause macromolecular poration in synthetic lipid vesicles at low peptide concentration and at neutral pH. These unique 26-residue peptides, which we call macrolittins, release macromolecules from lipid bilayer vesicles made from zwitterionic PC lipids at peptide to lipid ratios as low as 1:1000, a property that is almost unprecedented among known membrane permeabilizing peptides. The macrolittins exist as membrane-spanning α-helices. They cause dramatic bilayer thinning and form large pores in planar supported bilayers. The high potency of these peptides is likely due to their ability to stabilize bilayer edges by a process that requires specific electrostatic interactions between peptides.

Reported serial positions of true and illusory memories in the Deese/Roediger/McDermott paradigm.

One of the easiest ways to induce illusory memories in the laboratory is to use the Deese-Roediger-McDermott (DRM) word-list paradigm. Researchers have used this paradigm not only to study people's memories of stimuli that were not actually presented, but also to study the phenomenological qualities of these illusions. In four experiments, the current investigation explored a phenomenological quality of illusory memories that has received almost no attention, specifically, temporality. A serial position task was incorporated into the DRM paradigm to examine temporal attributes of participants' true and false memories. Effects of list strength, presentation order, and types of warnings were examined. Results showed consistent serial position responses for true and false memories. However, only responses for illusory memories were affected by manipulations at study. The current findings thus lend support to encoding-based explanations of false recollections.