The α-(1,3)-glucan synthase gene agsE impacts the secretome of Aspergillus niger.

Aspergillus niger is widely used as a cell factory for the industrial production of enzymes. Previously, it was shown that deletion of α-1-3 glucan synthase genes results in smaller micro-colonies in liquid cultures of Aspergillus nidulans. Also, it has been shown that small wild-type Aspergillus niger micro-colonies secrete more protein than large mirco-colonies.

Heterogeneity in Spore Aggregation and Germination Results in Different Sized, Cooperative Microcolonies in an Aspergillus niger Culture.

The fungus Aspergillus niger is among the most abundant fungi in the world and is widely used as a cell factory for protein and metabolite production. This fungus forms asexual spores called conidia that are used for dispersal. Notably, part of the spores and germlings aggregate in an aqueous environment.

Quantifying Positional Isomers (QPI) by Top-Down Mass Spectrometry.

Proteomics has exposed a plethora of posttranslational modifications, but demonstrating functional relevance requires new approaches. Top-down proteomics of intact proteins has the potential to fully characterize protein modifications in terms of amount, site(s), and the order in which they are deposited on the protein; information that so far has been elusive to extract by shotgun proteomics. Data acquisition and analysis of intact multimodified proteins have however been a major challenge, in particular for positional isomers that carry the same number of modifications at different sites.

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons.

The differentiation of neuronal stem cells into polarized neurons is a well-coordinated process which has mostly been studied in classical non-human model systems, but to what extent these findings are recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured hiPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. The neuron transcriptome and proteome shows extensive remodeling, with differential expression profiles of ~1100 transcripts and ~2200 proteins during neuronal differentiation and polarization.

Cortical anchoring of the microtubule cytoskeleton is essential for neuron polarity.

The development of a polarized neuron relies on the selective transport of proteins to axons and dendrites. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is poorly understood. Using the in vivo model system , we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33).

Feedback-Driven Assembly of the Axon Initial Segment.

The axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood.

Conserved crosstalk between histone deacetylation and H3K79 methylation generates DOT1L-dose dependency in HDAC1-deficient thymic lymphoma.

DOT1L methylates histone H3K79 and is aberrantly regulated in MLL-rearranged leukemia. Inhibitors have been developed to target DOT1L activity in leukemia, but cellular mechanisms that regulate DOT1L are still poorly understood. We have identified the histone deacetylase Rpd3 as a negative regulator of budding yeast Dot1.

MAP7 family proteins regulate kinesin-1 recruitment and activation.

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1.

Regulation of KIF1A-Driven Dense Core Vesicle Transport: Ca/CaM Controls DCV Binding and Liprin-α/TANC2 Recruits DCVs to Postsynaptic Sites.

Tight regulation of neuronal transport allows for cargo binding and release at specific cellular locations. The mechanisms by which motor proteins are loaded on vesicles and how cargoes are captured at appropriate sites remain unclear. To better understand how KIF1A-driven dense core vesicle (DCV) transport is regulated, we identified the KIF1A interactome and focused on three binding partners, the calcium binding protein calmodulin (CaM) and two synaptic scaffolding proteins: liprin-α and TANC2.

A System-wide Approach to Monitor Responses to Synergistic BRAF and EGFR Inhibition in Colorectal Cancer Cells.

Intrinsic and/or acquired resistance represents one of the great challenges in targeted cancer therapy. A deeper understanding of the molecular biology of cancer has resulted in more efficient strategies, where one or multiple drugs are adopted in novel therapies to tackle resistance. This beneficial effect of using combination treatments has also been observed in colorectal cancer patients harboring the BRAF(V600E) mutation, whereby dual inhibition of BRAF(V600E) and EGFR increases antitumor activity.

Actin from the apicomplexan Neospora caninum (NcACT) has different isoforms in 2D electrophoresis.

Apicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum - a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique.

H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2.

The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle.

Endogenous androgen receptor proteomic profiling reveals genomic subcomplex involved in prostate tumorigenesis.

Androgen receptor (AR) is a key player in prostate cancer development and progression. Here we applied immunoprecipitation mass spectrometry of endogenous AR in LNCaP cells to identify components of the AR transcriptional complex. In total, 66 known and novel AR interactors were identified in the presence of synthetic androgen, most of which were critical for AR-driven prostate cancer cell proliferation.

Erratum: Microtubule minus-end regulation at spindle poles by an ASPM-katanin complex.

This corrects the article DOI: 10.1038/ncb3511.

Microtubule minus-end regulation at spindle poles by an ASPM-katanin complex.

ASPM (known as Asp in fly and ASPM-1 in worm) is a microcephaly-associated protein family that regulates spindle architecture, but the underlying mechanism is poorly understood. Here, we show that ASPM forms a complex with another protein linked to microcephaly, the microtubule-severing ATPase katanin. ASPM and katanin localize to spindle poles in a mutually dependent manner and regulate spindle flux.

The atheroma plaque secretome stimulates the mobilization of endothelial progenitor cells ex vivo.

Endothelial progenitor cells (EPCs) constitute a promising alternative in cardiovascular regenerative medicine due to their assigned role in angiogenesis and vascular repair. In response to injury, EPCs promote vascular remodeling by replacement of damaged endothelial cells and/or by secreting angiogenic factors over the damaged tissue. Nevertheless, such mechanisms need to be further characterized.

Quantitative Map of Proteome Dynamics during Neuronal Differentiation.

Neuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hippocampal neurons at different developmental stages and monitored changes in protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Almost one third of all 4,500 proteins quantified underwent a more than 2-fold expression change during neuronal differentiation, indicating extensive remodeling of the neuron proteome.

Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X) and (X)K/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics.

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end.

Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands.

Membrane-Depolarizing Channel Blockers Induce Selective Glioma Cell Death by Impairing Nutrient Transport and Unfolded Protein/Amino Acid Responses.

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca and K channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown.

Association of Cell Adhesion Molecules Contactin-6 and Latrophilin-1 Regulates Neuronal Apoptosis.

In view of important neurobiological functions of the cell adhesion molecule contactin-6 (Cntn6) that have emerged from studies on null-mutant mice and autism spectrum disorders patients, we set out to examine pathways underlying functions of Cntn6 using a proteomics approach. We identified the cell adhesion GPCR latrophilin-1 (Lphn1, a.k.

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1.

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants.

Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome.

Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, autoinhibited and an open, active conformation. Although the open conformation of mDia2 induces actin assembly thereby controlling many cellular processes, mDia2 possesses also actin-independent and conformation-insensitive scaffolding roles related to microtubules and p53, respectively. Thus, we hypothesize that mDia2 may have other unappreciated functions and regulatory modes.

Molecular Pathway of Microtubule Organization at the Golgi Apparatus.

The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering.