protaTETHER - A Method for the Incorporation of Variable Linkers in Protein Fusions Reveals Impacts of Linker Flexibility in a PKAc-GFP Fusion Protein

Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemed protaTETHER, utilizes reiterative oligomer design, PCR-mediated linker library generation, and restriction enzyme-free cloning methods in a straightforward, three-step cloning process. We utilize a fusion between the catalytic subunit of cAMP-dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHER method, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc-GFP fusions, and use these results to select a PKAc-GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression

A scalable optical detection scheme for matter wave interferometry

Imaging of surface adsorbed molecules is investigated as a novel detection method for matter wave interferometry with fluorescent particles. Mechanically magnified fluorescence imaging turns out to be an excellent tool for recording quantum interference patterns. It has a good sensitivity and yields patterns of high visibility. The spatial resolution of this technique is only determined by the Talbot gratings and can exceed the optical resolution limit by an order of magnitude. A unique advantage of this approach is its scalability: for certain classes of nano-sized objects, the detection sensitivity will even increase significantly with increasing size of the particle.Comment: 10 pages, 4 figure

Simultaneous whole-animal 3D-imaging of neuronal activity using light field microscopy

3D functional imaging of neuronal activity in entire organisms at single cell level and physiologically relevant time scales faces major obstacles due to trade-offs between the size of the imaged volumes, and spatial and temporal resolution. Here, using light-field microscopy in combination with 3D deconvolution, we demonstrate intrinsically simultaneous volumetric functional imaging of neuronal population activity at single neuron resolution for an entire organism, the nematode Caenorhabditis elegans. The simplicity of our technique and possibility of the integration into epi-fluoresence microscopes makes it an attractive tool for high-speed volumetric calcium imaging.Comment: 25 pages, 7 figures, incl. supplementary informatio

Dimensionality and dynamics in the behavior of C. elegans

A major challenge in analyzing animal behavior is to discover some underlying simplicity in complex motor actions. Here we show that the space of shapes adopted by the nematode C. elegans is surprisingly low dimensional, with just four dimensions accounting for 95% of the shape variance, and we partially reconstruct "equations of motion" for the dynamics in this space. These dynamics have multiple attractors, and we find that the worm visits these in a rapid and almost completely deterministic response to weak thermal stimuli. Stimulus-dependent correlations among the different modes suggest that one can generate more reliable behaviors by synchronizing stimuli to the state of the worm in shape space. We confirm this prediction, effectively "steering" the worm in real time.Comment: 9 pages, 6 figures, minor correction

Biology of the Mi-2/NuRD Complex in SLAC (Stemness, Longevity/Ageing, and Cancer)

The dynamic chromatin activities of Mi-2/Nucleosome Remodeling and Histone deacetylation (Mi-2/NuRD) complexes in mammals are at the basis of current research on stemness, longevity/ageing, and cancer (4-2-1/SLAC), and have been widely studied over the past decade in mammals and the elegant model organism, Caenorhabditis elegans. Interestingly, a common emergent theme from these studies is that of distinct coregulator-recruited Mi-2/NuRD complexes largely orchestrating the 4-2-1/SLAC within a unique paradigm by maintaining genome stability via DNA repair and controlling three types of transcriptional programs in concert in a number of cellular, tissue, and organism contexts. Thus, the core Mi-2/NuRD complex plays a central role in 4-2-1/SLAC. The plasticity and robustness of 4-2-1/SLAC can be interpreted as modulation of specific coregulator(s) within cell-specific, tissue-specific, stage-specific, or organism-specific niches during stress induction, ie, a functional module and its networking, thereby conferring differential responses to different environmental cues. According to “Occam’s razor”, a simple theory is preferable to a complex one, so this simplified notion might be useful for exploring 4-2-1/SLAC with a holistic view. This thought could also be valuable in forming strategies for future research, and could open up avenues for cancer prevention and antiageing strategies

Optical Silencing of C. elegans Cells with Arch Proton Pump

BACKGROUND: Optogenetic techniques using light-driven ion channels or ion pumps for controlling excitable cells have greatly facilitated the investigation of nervous systems in vivo. A model organism, C. elegans, with its small transparent body and well-characterized neural circuits, is especially suitable for optogenetic analyses. METHODOLOGY/PRINCIPAL FINDINGS: We describe the application of archaerhodopsin-3 (Arch), a recently reported optical neuronal silencer, to C. elegans. Arch::GFP expressed either in all neurons or body wall muscles of the entire body by means of transgenes were localized, at least partially, to the cell membrane without adverse effects, and caused locomotory paralysis of worms when illuminated by green light (550 nm). Pan-neuronal expression of Arch endowed worms with quick and sustained responsiveness to such light. Worms reliably responded to repeated periods of illumination and non-illumination, and remained paralyzed under continuous illumination for 30 seconds. Worms expressing Arch in different subsets of motor neurons exhibited distinct defects in the locomotory behavior under green light: selective silencing of A-type motor neurons affected backward movement while silencing of B-type motor neurons affected forward movement more severely. Our experiments using a heat-shock-mediated induction system also indicate that Arch becomes fully functional only 12 hours after induction and remains functional for more than 24 hour. CONCLUSIONS/SGNIFICANCE: Arch can be used for silencing neurons and muscles, and may be a useful alternative to currently widely used halorhodopsin (NpHR) in optogenetic studies of C. elegans

Mesoscopic organization reveals the constraints governing C. elegans nervous system

One of the biggest challenges in biology is to understand how activity at the cellular level of neurons, as a result of their mutual interactions, leads to the observed behavior of an organism responding to a variety of environmental stimuli. Investigating the intermediate or mesoscopic level of organization in the nervous system is a vital step towards understanding how the integration of micro-level dynamics results in macro-level functioning. In this paper, we have considered the somatic nervous system of the nematode Caenorhabditis elegans, for which the entire neuronal connectivity diagram is known. We focus on the organization of the system into modules, i.e., neuronal groups having relatively higher connection density compared to that of the overall network. We show that this mesoscopic feature cannot be explained exclusively in terms of considerations, such as optimizing for resource constraints (viz., total wiring cost) and communication efficiency (i.e., network path length). Comparison with other complex networks designed for efficient transport (of signals or resources) implies that neuronal networks form a distinct class. This suggests that the principal function of the network, viz., processing of sensory information resulting in appropriate motor response, may be playing a vital role in determining the connection topology. Using modular spectral analysis, we make explicit the intimate relation between function and structure in the nervous system. This is further brought out by identifying functionally critical neurons purely on the basis of patterns of intra- and inter-modular connections. Our study reveals how the design of the nervous system reflects several constraints, including its key functional role as a processor of information.Comment: Published version, Minor modifications, 16 pages, 9 figure