A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits.

Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.

CryoET of ß-amyloid and tau within postmortem Alzheimer's disease brain.

A defining pathological feature of most neurodegenerative diseases is the assembly of proteins into amyloid that form disease-specific structures1. In Alzheimer's disease, this is characterized by the deposition of ß-amyloid and tau with disease-specific conformations. The in situ structure of amyloid in the human brain is unknown. Here, using cryo-fluorescence microscopy-targeted cryo-sectioning, cryo-focused ion beam-scanning electron microscopy lift-out and cryo-electron tomography, we determined in-tissue architectures of ß-amyloid and tau pathology in a postmortem Alzheimer's disease donor brain. ß-amyloid plaques contained a mixture of fibrils, some of which were branched, and protofilaments, arranged in parallel arrays and lattice-like structures. Extracellular vesicles and cuboidal particles defined the non-amyloid constituents of ß-amyloid plaques. By contrast, tau inclusions formed parallel clusters of unbranched filaments. Subtomogram averaging a cluster of 136 tau filaments in a single tomogram revealed the polypeptide backbone conformation and filament polarity orientation of paired helical filaments within tissue. Filaments within most clusters were similar to each other, but were different between clusters, showing amyloid heterogeneity that is spatially organized by subcellular location. The in situ structural approaches outlined here for human donor tissues have applications to a broad range of neurodegenerative diseases.

Multidimensional Dynamics of the Proteome in the Neurodegenerative and Aging Mammalian Brain.

The amount of any given protein in the brain is determined by the rates of its synthesis and destruction, which are regulated by different cellular mechanisms. Here, we combine metabolic labeling in live mice with global proteomic profiling to simultaneously quantify both the flux and amount of proteins in mouse models of neurodegeneration. In multiple models, protein turnover increases were associated with increasing pathology. This method distinguishes changes in protein expression mediated by synthesis from those mediated by degradation. In the AppNL-F knockin mouse model of Alzheimer's disease, increased turnover resulted from imbalances in both synthesis and degradation, converging on proteins associated with synaptic vesicle recycling (Dnm1, Cltc, Rims1) and mitochondria (Fis1, Ndufv1). In contrast to disease models, aging in wild-type mice caused a widespread decrease in protein recycling associated with a decrease in autophagic flux. Overall, this simple multidimensional approach enables a comprehensive mapping of proteome dynamics and identifies affected proteins in mouse models of disease and other live animal test settings.

Iminoboranes With Parent B=NH Entity: Imino Group Metathesis, Nucleophilic Reactivity and N-N Coupling.

While R2 C=N-R double bonds in organic imines are well established, the related iminoboranes R-B=N-R are either prone to oligomerization or can only be stabilized at sufficient steric congestion. In particular, the examples of unsubstituted parent B=N-H entity are rare. We demonstrate that the amino imidazoline-2-imine ligand system (HAmIm) not only gives rise to the isolation of a parent (AmIm)B=N-H iminoborane, but also to species of type (AmIm)B=N-SiMe3 with concomitant stabilization by lithium bromide. The double bond character in these systems is unambiguously corroborated by DFT calculations. The steric accessibility of the (AmIm)B=NH unit allows facile reactivity including metathesis reactions with C=O and C=S bonds, nucleophilic addition toward organic and organometallic carbonyl compounds, but also oxidative N-N coupling within a dimeric unit. Thus, the chemical behavior of the (AmIm)B=N-H and (AmIm)B=N-SiMe3 is distinctly different from that of organic imines.

Reactions of a Four-Membered Borete with Carbon, Silicon, and Gallium Donor Ligands: Fused and Spiro-Type Boracycles.

Donor-acceptor cyclopropanes or cyclobutanes are dipolar reagents, which are widely used in the synthesis of complex organic (hetero)cycles in ring expansion reactions. Applying this concept to boron containing heterocycles, the four-membered borete cyclo-iPr2 N-BC10 H6 reacted with the carbon donor ligands 2,6-xylylisonitrile and the carbene IMes :C(NMesCH)2 with ring expansion and ring fusion, respectively. In particular, the tetracyclic structure formed with IMes displays zwitterionic character and absorption in the visible region. In contrast to the carbene IMes, the heavier carbenoids :Si(NDippCH)2 and :Ga(AmIm) with a two-coordinate donor atom afford spiro-type bicyclic compounds, which display four-coordinate geometry at silicon or gallium. (TD-)DFT calculations provide deeper insight into the mechanism of formation and the absorption properties of these new compounds.

Bridging length scales from molecules to the whole organism by cryoCLEM and cryoET.

Resolving atomic structures of isolated proteins has uncovered mechanisms and fundamental processes in biology. However, many functions can only be tested in the context of intact cells and tissues that are many orders of magnitude larger than the macromolecules on which they depend. Therefore, methods that interrogate macromolecular structure in situ provide a means of directly relating structure to function across length scales. Here, we developed several workflows using cryogenic correlated light and electron microscopy (cryoCLEM) and electron tomography (cryoET) that can bridge this gap to reveal the molecular infrastructure that underlies higher order functions within cells and tissues. We also describe experimental design considerations, including cryoCLEM labelling, sample preparation, and quality control, for determining the in situ molecular architectures within native, hydrated cells and tissues.

Reductive Al-B σ-Bond Formation in Alumaboranes: Facile Scission of Polar Multiple Bonds.

We present facile access to an alumaborane species with electron precise Al-B σ-bond. The reductive rearrangement of 1-(AlI2 ), 8-(BMes2 ) naphthalene (Mes=2,4,6-Me3 C6 H2 ) affords the alumaborane species cyclo-(1,8-C10 H6 )-[1-Al(Mes)(OEt2 )-8-B(Mes)] with a covalent Al-B σ-bond. The Al-B σ-bond performs the reductive scission of multiple bonds: S=C(NiPrCMe)2 affords the naphthalene bridged motif B-S-Al(NHC), NHC=N-heterocyclic carbene, while O=CPh2 is deoxygenated to afford an B-O-Al bridged species with incorporation of the remaining ≡CPh2 fragment into the naphthalene scaffold. The reaction with isonitrile Xyl-N≡C (Xyl=2,6-Me2 C6 H4 ) proceeds via a proposed (amino boryl) carbene species; which adds a second equivalent of isonitrile to ultimately form the Al-N-B bridged species cyclo-(1,8-C10 H6 )-[1-Al(Mes)-N(Xyl)-8-B{C(Mes)=C-N-Xyl}] with complete scission of the C≡N triple bond. The latter reaction is supported with isolated intermediates and by DFT calculations.

Superseding ß-Diketiminato Ligands: An Amido Imidazoline-2-Imine Ligand Stabilizes the Exhaustive Series of B=X Boranes (X=O, S, Se, Te).

Boron reluctantly forms B=X (X=O, S, Se, Te) moieties, which has stimulated the quest for such species in the past few years. Based on the N,N'-chelating ß-diketiminato ligand (HNacNac), a new amido imidazoline-2-imine ligand system (HAmIm) is presented, giving rise to the isolation of an exhaustive series of Lewis acid free, monomeric chalcogen B=X boranes with documented π-bond character between boron and the chalcogen. The chalcogenoboranes are isoelectronic and isolobal to the respective ketones. The chemical behavior of the oxoborane (B=O) strongly resembles the classical carbonyl reactivity in C=O bonds. The improved stability provided by HAmIm arises from the formation of more-stable five-membered boron chelates versus the six-membered NacNac analogues and from the imidazoline-2-imine moiety providing enhanced σ- and π-donation. The HAmIm ligand class may supersede the widely employed NacNac system in certain applications.

Two π-Electrons Make the Difference: From BODIPY to BODIIM Switchable Fluorescent Dyes.

(aza-)BODIPY dyes (boron dipyrromethene dyes) are well-established fluorophores due to their large quantum yields, stability, and diversity, which led to promising applications including imaging techniques, sensors, organic (opto)electronic materials, or biomedical applications. Although the control of the optical properties in (aza-)BODIPY dyes by peripheral functional groups is well studied, we herein present a novel approach to modify the 12 π-electron core of the dipyrromethene scaffold. The replacement of two carbon atoms in the ß-position of a BODIPY dye by two nitrogen atoms afforded a 14 π-electron system, which was termed BODIIM (boron diimidazolylmethene) in systematic analogy to the BODIPY dyes. Remarkably, the BODIIM dye was obtained with a BH2 -rigidifying entity, which is currently elusive and highly sought after for the BODIPY dye class. DFT-Calculations confirm the [12+2] π-electron relationship between BODIPY and BODIIM and reveal a strong shape correlation between LUMO in the BODIPY and the HOMO of the BODIIM. The modification of the π-system leads to a dramatic shift of the optical properties, of which the fluorescent emission is most noteworthy and occurs at much larger Stokes shift, that is, ≈500 cm-1 in BODIPY versus >4170 cm-1 in BODIIM system in all solvents investigated. Nucleophilic reactivity was found at the meso-carbon atom in the formation of stable borane adducts with a significant shift of the fluorescent emission, and this behavior contrasts the reactivity of conventional BODIPY systems. In addition, the reverse decomplexation of the borane adducts was demonstrated in reactions with a representative N-heterocyclic carbene to retain the strongly fluorescent BODIIM compound, which suggests applications as fully reversible fluorescent switch.

Benzyl Borane NHC Adducts: Beyond B-C Bond Scission.

Benzyl-substituted boronates and borates are widely employed as mild sources in radical or anionic transfer reactions of benzyl entities. In this process the B-C bond to the benzyl moiety is essentially ruptured. In contrast, reactions with retention of the B-C bond are poorly investigated although several other reactive sites in benzyl-boron systems are clearly inherent. In this respect, the novel reactivity of the representative borane adduct IiPr-BH2 Bn [IiPr=:C{N(iPr)CH}2 , Bn=CH2 C6 H5 ] is demonstrated. Dihalogenation of the BH2 entity is observed with BCl3 and BBr3 , whereas BI3 either affords IiPr-BHI2 or proceeds with borylation of the aromatic phenyl ring to give a hydride-bridged bisborylated species. The photochemical mono- and dihalogenation of the benzylic CH2 group was demonstrated with elemental bromine Br2 . The brominated product IiPr-BBr2 -CHBr-C6 H5 was borylated at the benzylic carbon atom in an umpolung event with BI3 to afford the zwitterion IiPr-BI-CH(BI3 )-C6 H5 .

N-Heterocyclic Carbene Adducts of Alkynyl Functionalized 1,3,2-Dithioborolanes.

Alkynyl functionalized boron compounds are versatile intermediates in the areas of medicinal chemistry, materials science, and optical materials. In particular, alkynyl boronate esters [R1-C≡C-B(OR2)2] are of interest since they provide reactivity at both the alkyne entity, with retention of the B-C bond or alkyne transfer to electrophilic substrates with scission of the latter. The boron atom is commonly well stabilized due to (i) the extraordinary strength of two B-O bonds, and (ii) the chelate effect exerted by a bifunctional alcohol. We reasoned that the replacement of a B-O for a B-S bond would lead to higher reactivity and post-functionalization in the resulting alkynyl boronate thioesters [R1-C≡C-B(S2X)]. Access to this poorly investigated class of compounds starts form chloro dithioborolane cyclo-Cl-B(S2C2H4) as a representative example. Whereas syntheses of three coordinate alkynyl boronate thioesters [R1-C≡C-B(S2X)] proved to be ineffective, the reactions of NHC-adducts (NHC = N-heterocyclic carbene) of cyclo-Cl-B(S2C2H4) afforded the alkyne substituted thioboronate esters in good yield. The products NHC-B(S2C2H4)(C≡C-R1) are remarkably stable towards water and air, which suggests their use as boron-based building blocks for applications akin to oxygen-based boronate esters.

Hierarchical organization and genetically separable subfamilies of PSD95 postsynaptic supercomplexes.

PSD95 is an abundant postsynaptic scaffold protein in glutamatergic synapses that assembles into supercomplexes composed of over 80 proteins including neurotransmitter receptors, ion channels and adhesion proteins. How these diverse constituents are organized into PSD95 supercomplexes in vivo is poorly understood. Here, we dissected the supercomplexes in mice combining endogenous gene-tagging, targeted mutations and quantitative biochemical assays. Generating compound heterozygous mice with two different gene-tags, one on each Psd95 allele, showed that each ~1.5 MDa PSD95-containing supercomplex contains on average two PSD95 molecules. Gene-tagging the endogenous GluN1 and PSD95 with identical Flag tags revealed N-methyl D-aspartic acid receptors (NMDARs) containing supercomplexes that represent only 3% of the total population of PSD95 supercomplexes, suggesting there are many other subtypes. To determine whether this extended population of different PSD95 supercomplexes use genetically defined mechanisms to specify their assembly, we tested the effect of five targeted mouse mutations on the assembly of known PSD95 interactors, Kir2.3, Arc, IQsec2/BRAG1 and Adam22. Unexpectedly, some mutations were highly selective, whereas others caused widespread disruption, indicating that PSD95 interacting proteins are organized hierarchically into distinct subfamilies of ~1.5 MDa supercomplexes, including a subpopulation of Kir2.3-NMDAR ion channel-channel supercomplexes. Kir2.3-NMDAR ion channel-channel supercomplexes were found to be anatomically restricted to particular brain regions. These data provide new insight into the mechanisms that govern the constituents of postsynaptic supercomplexes and the diversity of synapse types. Read the Editorial Highlight for this article on page 500. Cover Image for this issue: doi. 10.1111/jnc.13811.

Charge-Compensated Metallacarborane Building Blocks for Conjugation with Peptides.

The cobalt bis(dicarbollide) complex [commo-3,3'-Co(1,2-C2 B9 H11 )2 ](-) has captured much attention in biochemical and medical contexts, in particular for the treatment of tumors by boron neutron capture therapy (BNCT). Derivatives of cobalt bis(dicarbollide) are commonly prepared through ring-opening reactions of cyclic oxonium ions, so the corresponding products are usually charged. Furthermore, attempts to incorporate cobalt bis(dicarbollide) into peptides are rare, despite obvious potential advantages. Here the synthesis of an imidazolium-based charge-compensated cobalt bis(dicarbollide) building block, which allows additional modifications with moieties of biochemical relevance, such as monosaccharides, is reported. Furthermore, conjugates of these building blocks with the Y1 -receptor-selective derivative of neuropeptide Y ([F(7) ,P(34) ]-NPY) retained excellent response to hY1 receptors found to be overexpressed in breast tumors and metastases.

Cobalt Boryl Complexes: Enabling and Exploiting Migratory Insertion in Base-Metal-Mediated Borylation.

Cobalt boryl complexes, which have only been sporadically reported, can be accessed systematically with remarkable (but controllable) variation in the nature of the M-B bond. Complexes incorporating a very strong trans σ-donor display unparalleled inertness, reflected in retention of the M-B bond even in the presence of extremely strong acid. By contrast, the use of the strong π-acceptor CO in the trans position, results in significant Co-B elongation and to labilization of the boryl ligand via unprecedented CO migratory insertion. Such chemistry provides a pathway for the generation of coordinative unsaturation, thereby enabling ligand substitution and/or substrate assimilation. Alkene functionalization by boryl transfer, a well-known reaction for noble metals such as Rh or Pt, can thus be effected by an 18-electron base-metal complex.

Circumventing redox chemistry: synthesis of transition metal boryl complexes from a boryl nucleophile by decarbonylation.

The very strong reducing capabilities of the boryllithium nucleophile (THF)2Li{B(NDippCH)2} (1, Dipp = 2,6-iPr2C6H3) render impractical its use for the direct introduction of the {B(NDippCH)2} ligand via metathesis chemistry into the immediate coordination sphere of transition metals (d(n), with n ≠ 0 or 10). Instead, 1 typically reacts with metal halide, amide and hydrocarbyl electrophiles either via electron transfer or halide abstraction. Evidence for the formation of M-B bonds is obtained only in the case of the d(5) system [{(HCDippN)2B}Mn(THF)(µ-Br)]2. Lower oxidation state metal carbonyl complexes such as Fe(CO)5 and Cr(CO)6 react with 1 via nucleophilic attack at the carbonyl carbon atom to give boryl-functionalized Fischer carbene complexes Fe(CO)4{C(OLi(THF)3)B(NDippCH)2} and Cr(CO)5{C(OLi(THF)2)B(NDippCH)2}. Although C-to-M boryl transfer does not occur for these formally anionic systems, more labile charge neutral bora-acyl derivatives of the type LnM{C(O)B(NDippCH)2} [LnM = Mn(CO)5, Re(CO)5, CpFe(CO)2] can be synthesized, which cleanly lose CO to generate M-B bonds. From a mechanistic standpoint, an archetypal organometallic mode of reactivity, carbonyl extrusion, has thus been shown to be applicable to the boryl ligand class, with (13)C isotopic labeling studies confirming a dissociation/migration pathway. These proof-of-methodology synthetic studies can be extended beyond boryl complexes of the group 7 and 8 metals (for which a number of versatile synthetic routes already exist) to provide access to complexes of cobalt, which have hitherto proven only sporadically accessible.

Electrophile-induced nucleophilic substitution of the nido-dicarbaundecaborate anion nido-7,8-C2B9H12- by conjugated heterodienes.

Substitution of the dicarbaundecaborate anion nido-7,8-C2B9H12(-) (1) by precise hydride abstraction followed by nucleophilic attack usually leads to symmetric products 10-R-nido-7,8-C2B9H11. However, thioacetamide (MeC(S)NH2) as nucleophile and acetone/AlCl3 as hydride abstractor gave asymmetric 9-[MeC(NHiPr)S]-nido-7,8-C2B9H11 (2), whereas N,N-dimethylthioacetamide (MeC(S)NMe2) gave the expected symmetric 10-[MeC(NMe2)S]-nido-7,8-C2B9H11 (4). For the formation of 2, acetone and thioacetamide are assumed to give the intermediate MeC(S)N(CMe2) (3), which then attacks 1 with formation of 2. Similarly, reaction of acetyliminium chloride [MeC(O)NH(CPh2)]Cl (5) with 1 in THF gave a mixture of 9- and 10-substituted [MeC(NHCHPh2)O]-nido-7,8-C2B9H11 (6 and 7, respectively). These reactions are the first examples in which compounds (here heterodienes) that unite the functionalities of both hydride acceptor and nucleophilic site react with 1 in a bimolecular fashion. Furthermore, the analogous reaction of 1 and 5 (in an equilibrium mixture with acetyl chloride and benzophenone imine) in MeCN afforded 10-[MeC(NCPh2)NH]-nido-7,8-C2B9H11 (8) and MeC(O)NHCHPh2 (9).