Adam Williamson

Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex.

The anaphase-promoting complex (APC/C) orchestrates progression through mitosis by decorating cell-cycle regulators with ubiquitin chains. To nucleate chains, the APC/C links ubiquitin to a lysine in substrates, but to elongate chains it modifies lysine residues in attached ubiquitin moieties. The mechanism enabling the APC/C, and ubiquitin ligases in general, to switch from lysine residues in substrates to specific ones in ubiquitin remains poorly understood. Here, we determine the topology and the mechanism of assembly for the ubiquitin chains mediating functions of the human APC/C. We find that the APC/C triggers substrate degradation by assembling K11-linked ubiquitin chains, the efficient formation of which depends on a surface of ubiquitin, the TEK-box. Strikingly, homologous TEK-boxes are found in APC/C substrates, where they facilitate chain nucleation. We propose that recognition of similar motifs in substrates and ubiquitin enables the APC/C to assemble ubiquitin chains with the specificity and efficiency required for tight cell-cycle control.

Identification of a physiological E2 module for the human anaphase-promoting complex

Ubiquitination by the anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The human APC/C promotes the degradation of mitotic regulators by assembling K11-linked ubiquitin chains, the formation of which is initiated by its E2 UbcH10. Here, we identify the conserved Ube2S as a K11-specific chain elongating E2 for human and Drosophila APC/C. Ube2S depends on the cell cycle-dependent association with the APC/C activators Cdc20 and Cdh1 for its activity. While depletion of Ube2S already inhibits APC/C in cells, the loss of the complete UbcH10/Ube2S-module leads to dramatic stabilization of APC/C substrates, severe spindle defects, and a strong mitotic delay. Ube2S and UbcH10 are tightly co-regulated in the cell cycle by APC/C-dependent degradation. We conclude that UbcH10 and Ube2S constitute a physiological E2-module for APC/C, the activity of which is required for spindle assembly and cell division.

High-performance transistors for bioelectronics through tuning of channel thickness

Transistors with tunable transconductance allow high-quality recordings of human brain rhythms. Despite recent interest in organic electrochemical transistors (OECTs), sparked by their straightforward fabrication and high performance, the fundamental mechanism behind their operation remains largely unexplored. OECTs use an electrolyte in direct contact with a polymer channel as part of their device structure. Hence, they offer facile integration with biological milieux and are currently used as amplifying transducers for bioelectronics. Ion exchange between electrolyte and channel is believed to take place in OECTs, although the extent of this process and its impact on device characteristics are still unknown. We show that the uptake of ions from an electrolyte into a film of poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) leads to a purely volumetric capacitance of 39 F/cm3. This results in a dependence of the transconductance on channel thickness, a new degree of freedom that we exploit to demonstrate high-quality recordings of human brain rhythms. Our results bring to the forefront a transistor class in which performance can be tuned independently of device footprint and provide guidelines for the design of materials that will lead to state-of-the-art transistor performance.

K11-linked ubiquitin chains as novel regulators of cell division.

Modification of proteins with ubiquitin chains is an essential regulatory event in cell cycle control. Differences in the connectivity of ubiquitin chains are believed to result in distinct functional consequences for the modified proteins. Among eight possible homogenous chain types, canonical Lys48-linked ubiquitin chains have long been recognized to drive the proteasomal degradation of cell cycle regulators, and Lys48 is the only essential lysine residue of ubiquitin in yeast. It thus came as a surprise that in higher eukaryotes atypical K11-linked ubiquitin chains regulate the substrates of the anaphase-promoting complex and control progression through mitosis. We discuss recent findings that shed light on the assembly and function of K11-linked chains during cell division.

Regulation of Ubiquitin Chain Initiation to Control the Timing of Substrate Degradation

Processive reactions, such as transcription or translation, often proceed through distinct initiation and elongation phases. The processive formation of polymeric ubiquitin chains can accordingly be catalyzed by specialized initiating and elongating E2 enzymes, but the functional significance for this division of labor has remained unclear. Here, we have identified sequence motifs in several substrates of the anaphase-promoting complex (APC/C) that are required for efficient chain initiation by its E2 Ube2C. Differences in the quality and accessibility of these chain initiation motifs can determine the rate of a substrates degradation without affecting its affinity for the APC/C, a mechanism used by the APC/C to control the timing of substrate proteolysis during the cell cycle. Based on our results, we propose that initiation motifs and their cognate E2s allow E3 enzymes to exert precise temporal control over substrate degradation.

Controlling Epileptiform Activity with Organic Electronic Ion Pumps

In treating epilepsy, the ideal solution is to act at a seizures onset, but only in the affected regions of the brain. Here, an organic electronic ion pump is demonstrated, which directly delivers on-demand pure molecules to specific brain regions. State-of-the-art organic devices and classical pharmacology are combined to control pathological activity in vitro, and the results are verified with electrophysiological recordings.

Localized Neuron Stimulation with Organic Electrochemical Transistors on Delaminating Depth Probes.

Organic electrochemical transistors are integrated on depth probes to achieve localized electrical stimulation of neurons. The probes feature a mechanical delamination process which leaves only a 4 μm thick film with embedded transistors inside the brain. This considerably reduces probe invasiveness and correspondingly improves future brain-machine interfaces.

Bioelectronic neural pixel: Chemical stimulation and electrical sensing at the same site

Significance Electronically and ionically conducting polymers provide a unique means to translate electronic addressing signals into chemically specific and spatiotemporally resolved delivery, without fluid flow. These materials have also been shown to provide high-fidelity electrophysiological recordings. Here, we demonstrate the combination of these qualities of organic electronics in multiple 20 × 20 µm delivery/sensing electrodes. The system is used to measure epileptic activity in a brain slice model, and to deliver inhibitory neurotransmitters to the same sites as the recordings. These results show that a single-cell-scale electrode has the ability to both record and chemically stimulate, demonstrating the local effects of therapeutic treatment, and opening a range of opportunities in basic neuroscience as well as medical technology development. Local control of neuronal activity is central to many therapeutic strategies aiming to treat neurological disorders. Arguably, the best solution would make use of endogenous highly localized and specialized regulatory mechanisms of neuronal activity, and an ideal therapeutic technology should sense activity and deliver endogenous molecules at the same site for the most efficient feedback regulation. Here, we address this challenge with an organic electronic multifunctional device that is capable of chemical stimulation and electrical sensing at the same site, at the single-cell scale. Conducting polymer electrodes recorded epileptiform discharges induced in mouse hippocampal preparation. The inhibitory neurotransmitter, γ-aminobutyric acid (GABA), was then actively delivered through the recording electrodes via organic electronic ion pump technology. GABA delivery stopped epileptiform activity, recorded simultaneously and colocally. This multifunctional “neural pixel” creates a range of opportunities, including implantable therapeutic devices with automated feedback, where locally recorded signals regulate local release of specific therapeutic agents.

The Colossus of ubiquitylation: decrypting a cellular code.

Ubiquitylation is an essential posttranslational modification that can regulate the stability, activity, and localization of thousands of proteins. The reversible attachment of ubiquitin as well as interpretation of the ubiquitin signal depends on dynamic protein networks that are challenging to analyze. In this perspective, we discuss tools of the trade that have recently been developed to dissect mechanisms of ubiquitin-dependent signaling, thereby revealing the critical features of an important cellular code.

Preparation of Synchronized Human Cell Extracts to Study Ubiquitination and Degradation

Ubiquitination and protein degradation regulate cell cycle progression in all eukaryotes. During mitosis, ubiquitination by the Anaphase-Promoting Complex/Cyclosome (APC/C) triggers sister chromatid separation and mitotic exit. The APC/C is tightly regulated by phosphorylation, ubiquitination, association of activators or inhibitors, and competitive binding of substrates. Much of our understanding of the mechanism of APC/C-dependent ubiquitination has been obtained from studies using extracts of Xenopus laevis eggs or synchronized human tissue culture cells. Here, we describe protocols to prepare extracts of synchronized human cells, and discuss experiments to use extracts for the biochemical analysis of APC/C-dependent ubiquitination.