Mechanical force induces mitochondrial fission.

Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria - via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces - results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm

Building ProteomeTools based on a complete synthetic human proteome.

We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of \u3e330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to \u3e1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange

Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype

qcML: an exchange format for quality control metrics from mass spectrometry experiments.

Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data. What has been missing, however, is a standard data exchange format for reporting these performance metrics. We therefore developed the qcML format, an XML-based standard that follows the design principles of the related mzML, mzIdentML, mzQuantML, and TraML standards from the HUPO-PSI (Proteomics Standards Initiative). In addition to the XML format, we also provide tools for the calculation of a wide range of quality metrics as well as a database format and interconversion tools, so that existing LIMS systems can easily add relational storage of the quality control data to their existing schema. We here describe the qcML specification, along with possible use cases and an illustrative example of the subsequent analysis possibilities. All information about qcML is available at http://code.google.com/p/qcml

New Tools for Engineered Networks of Neurons

The brain is a fascinating structure to investigate not only because of its staggering complexity but also because of its clinical importance. The human brain consists of more than 80 billion nerve cells, each receiving up to 10000 input connections. Despite big efforts in neuroscience, we still do not understand how the brain carries out its basic functions, such as information processing and storage, because the critical link between network structure and function remains largely unclear. The bottom-up neuroscience approach aims to answer these questions by engineering small, patterned networks of neurons in vitro. The complexity of the investigated system is thereby drastically reduced compared to top-down neuroscience approach, which examines the immensely complex brain as a whole. Advances in neuroscience have been driven by the development of new tools, such as the light microscope, patch-clamp, or more recently optogenetics. Here, the investigation of small neuronal networks is improved by developing new tools for culturing, measuring and stimulating neurons. First, the survival and function of neurons cultured in vitro at low densities are improved by suspending astrocytes cultured on cellulose-paper above the neurons. Astrocytes are a critical cell type in the brain and their addition in vitro creates a supportive and more physiological micro-environment, in particular for networks of low densities typically needed for bottom-up neuroscience. Astrocytic co-culture results in drastically improved neuronal viability and more frequent spontaneous spiking activity compared to mono-cultures. Second, the analysis of neuronal calcium imaging videos is simplified with an easy-to-use and robust computer program. Calcium imaging is a convenient way to measure the electrophysiological activity of neurons using fluorescence microscopy. Extracting the neuronal activity from these videos however is challenging due to the enormous amounts of data it generates. The developed tool allows to efficiently perform this repetitive task and frees up time for the actual analysis of the behavior of the recorded network of neurons. Third, the stimulation of neurons in vitro is improved with a local chemical stimulation platform based on the FluidFM, a force-controlled nanopipette. After gently approaching the FluidFM cantilever to the target neuron, the neurotransmitter glutamate is dispensed locally to stimulate the cell, thereby replicating the signal transmission between neurons. This platform is capable of reliably stimulating neurons with a control over the stimulation dose as can be measured electrically and optically on the level of both the stimulated neuron and the network. Finally, because the brain uses several different kinds of neurotransmitters and neuromodulators, it is advantageous to extend the FluidFM to contain multiple channels to allow it to release multiple compounds during an experiment. The required instrumentation for interfacing such novel cantilevers was developed and tested with prototypes for future developments. Overall, these techniques expand the toolkit for engineering and investigating networks of neurons and to improve our understanding of the brain

Force controlled SU-8 micropipettes fabricated with a sideways process

We established an in-plane microfabrication process of polymeric AFM micropipettes that include a microchannel with an aperture at the apex without further processing after wafer scale lithography. The channel was obtained by etching a sacrificial electrochemically plated copper layer. Such sideways fabrication scheme offers the possibility of designing various types of cantilevers with different shapes and lengths within a single wafer, such as a length of 500 µm or being connected by two triangular hollow tips. The probes were operated in optical beam deflection AFM mode. First, force-indentation curves were measured on substrates with different stiffness, and then microbeads were spatially manipulated in a hydrogel showing their reusability in case of clogging. Finally, we succeeded in using such probes to carry out cell-isolation experiments of neurons cultured in vitro (2D layers) and in hydrogels (3D architecture)

“Brains on a chip”: Towards engineered neural networks

The fundamental mechanisms of complex neural computation remain largely unknown, especially in respect to the characteristics of distinct neural circuits within the mammalian brain. The bottom-up approach of building well-defined neural networks with controlled topology has immense promise for improved reproducibility and increased target selectivity and response of drug action, along with hopes to unravel the relationships between functional connectivity and its imprinted physiological and pathological functions. In this review, we summarize the different approaches available for engineering neural networks treated analogously to a mathematical graph consisting of cell bodies and axons as nodes and edges, respectively. After discussing the advances and limitations of the current techniques in terms of cell placement to the nodes and guiding the growth of axons to connect them, the basic properties of patterned networks are analyzed in respect to cell survival and activity dynamics, and compared to that of in vivo and random in vitro cultures. Besides the fundamental scientific interest and relevance to drug and toxicology tests, we also visualize the possible applications of such engineered networks. The review concludes by comparing the possibilities and limitations of the different methods for realizing in vitro engineered neural networks in 2D

Mechanical force induces mitochondrial fission

Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria - via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces - results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm