Correction: Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

[This corrects the article DOI: 10.1371/journal.pone.0158295.].

Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges.

Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation.

Hundreds of L1CAM gene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditional L1CAM loss-of-function mutation and produced precisely matching control and L1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion of L1CAM dramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20-50% and ∼20-30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that the L1CAM mutation selectively decreased activity-dependent Na(+)-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations of L1CAM mutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.

Large-area molecular patterning with polymer pen lithography.

The challenge of constructing surfaces with nanostructured chemical functionality is central to many areas of biology and biotechnology. This protocol describes the steps required for performing molecular printing using polymer pen lithography (PPL), a cantilever-free scanning probe-based technique that can generate sub-100-nm molecular features in a massively parallel fashion. To illustrate how such molecular printing can be used for a variety of biologically relevant applications, we detail the fabrication of the lithographic apparatus and the deposition of two materials, an alkanethiol and a polymer onto a gold and silicon surface, respectively, and show how the present approach can be used to generate nanostructures composed of proteins and metals. Finally, we describe how PPL enables researchers to easily create combinatorial arrays of nanostructures, a powerful approach for high-throughput screening. A typical protocol for fabricating PPL arrays and printing with the arrays takes 48-72 h to complete, including two overnight waiting steps.

Scanning probe-enabled nanocombinatorics define the relationship between fibronectin feature size and stem cell fate.

We report the development of a powerful analytical method that utilizes a tilted elastomeric pyramidal pen array in the context of a scanning probe lithography experiment to rapidly prepare libraries having as many as 25 million features over large areas with a range of feature sizes from the nano- to microscale. This technique can be used to probe important chemical and biological processes, opening up the field of nanocombinatorics. In a proof-of-concept investigation of mesenchymal stem cell (MSC) differentiation, combinatorial patterns first enabled a rapid and systematic screening of MSC adhesion, as a function of feature size, while uniform patterns were used to study differentiation with statistically significant sample sizes. Without media containing osteogenic-inducing chemical cues, cells cultured on nanopatterned fibronectin substrates direct MSC differentiation towards osteogenic fates when compared to nonpatterned fibronectin substrates. This powerful and versatile approach enables studies of many systems spanning biology, chemistry, and engineering areas.

Positionally defined, binary semiconductor nanoparticles synthesized by scanning probe block copolymer lithography.

We report the first method for synthesizing binary semiconductor materials by scanning probe block copolymer lithography (SPBCL) in desired locations on a surface. In this work, we utilize SPBCL to create polymer features containing a desired amount of Cd(2+), which is defined by the feature volume. When they are subsequently reacted in H(2)S in the vapor phase, a single CdS nanoparticle is formed in each block copolymer (BCP) feature. The CdS nanoparticles were shown to be both crystalline and luminescent. Importantly, the CdS nanoparticle sizes can be tuned since their diameters depend on the volume of the originally deposited BCP feature.

Nanoreactors for studying single nanoparticle coarsening.

The ability to observe intermediate structures as part of coarsening processes that lead to the formation of single nanoparticles (NPs) is important in gaining fundamental insight pertaining to nanostructure growth. Here, we use scanning probe block copolymer lithography (SPBCL) to create "nanoreactors" having attoliter volumes, which confine Au NP nucleation and growth to features having diameters <150 nm on a substrate. With this technique, one can use in situ TEM to directly observe and study NP coarsening and differentiate Ostwald ripening from coalescence processes. Importantly, the number of metal atoms that can engage in coarsening can be controlled with this technique, and TEM "snapshots" of particle growth can be taken. The size of the resulting nanostructures can be controlled in the 2-10 nm regime.

Scanning probe block copolymer lithography.

Integration of individual nanoparticles into desired spatial arrangements over large areas is a prerequisite for exploiting their unique electrical, optical, and chemical properties. However, positioning single sub-10-nm nanoparticles in a specific location individually on a substrate remains challenging. Herein we have developed a unique approach, termed scanning probe block copolymer lithography, which enables one to control the growth and position of individual nanoparticles in situ. This technique relies on either dip-pen nanolithography (DPN) or polymer pen lithography (PPL) to transfer phase-separating block copolymer inks in the form of 100 or more nanometer features on an underlying substrate. Reduction of the metal ions via plasma results in the high-yield formation of single crystal nanoparticles per block copolymer feature. Because the size of each feature controls the number of metal atoms within it, the DPN or PPL step can be used to control precisely the size of each nanocrystal down to 4.8 ± 0.2 nm.

Beam pen lithography.

Lithography techniques are currently being developed to fabricate nanoscale components for integrated circuits, medical diagnostics and optoelectronics. In conventional far-field optical lithography, lateral feature resolution is diffraction-limited. Approaches that overcome the diffraction limit have been developed, but these are difficult to implement or they preclude arbitrary pattern formation. Techniques based on near-field scanning optical microscopy can overcome the diffraction limit, but they suffer from inherently low throughput and restricted scan areas. Highly parallel two-dimensional, silicon-based, near-field scanning optical microscopy aperture arrays have been fabricated, but aligning a non-deformable aperture array to a large-area substrate with near-field proximity remains challenging. However, recent advances in lithographies based on scanning probe microscopy have made use of transparent two-dimensional arrays of pyramid-shaped elastomeric tips (or 'pens') for large-area, high-throughput patterning of ink molecules. Here, we report a massively parallel scanning probe microscopy-based approach that can generate arbitrary patterns by passing 400-nm light through nanoscopic apertures at each tip in the array. The technique, termed beam pen lithography, can toggle between near- and far-field distances, allowing both sub-diffraction limit (100 nm) and larger features to be generated.

Nanoscale molecular transport: the case of dip-pen nanolithography.

In dip-pen nanolithography experiments, many groups have observed that different tips deliver the same ink at different rates. This article presents a quantitative model for understanding this phenomenon and, importantly, a way of controlling it. An inkjet printer is used to deliver controlled amounts of 16-mercaptohexadecanoic acid (MHA) to atomic force microscope tips in an array. Ink transport from each tip is studied as a function of the number of drops delivered. We show a nonlinear dependence of transport rates on the number of drops that arises from surface-area-dependent dissolution of MHA. From this work, MHA dissolution attempt frequencies were calculated to be between 1.3 x 10(9) and 4.4 x 10(9) Hz.

Polymer pen lithography.

We report a low-cost, high-throughput scanning probe lithography method that uses a soft elastomeric tip array, rather than tips mounted on individual cantilevers, to deliver inks to a surface in a "direct write" manner. Polymer pen lithography merges the feature size control of dip-pen nanolithography with the large-area capability of contact printing. Because ink delivery is time and force dependent, features on the nanometer, micrometer, and macroscopic length scales can be formed with the same tip array. Arrays with as many as about 11 million pyramid-shaped pens can be brought into contact with substrates and readily leveled optically to ensure uniform pattern development.