Mechanisms of Granulin Deficiency: Lessons from Cellular and Animal Models

The identification of causative mutations in the (pro)granulin gene (GRN) has been a major breakthrough in the research on frontotemporal dementia (FTD). So far, all FTD-associated GRN mutations are leading to neurodegeneration through a “loss-of-function” mechanism, encouraging researchers to develop a growing number of cellular and animal models for GRN deficiency. GRN is a multifunctional secreted growth factor, and loss of its function can affect different cellular processes. Besides loss-of-function (i.e., mostly premature termination codons) mutations, which cause GRN haploinsufficiency through reduction of GRN expression, FTD-associated GRN missense mutations have also been identified. Several of these missense mutations are predicted to increase the risk of developing neurodegenerative diseases through altering various key biological properties of GRN-like protein secretion, proteolytic processing, and neurite outgrowth. With the use of cellular and animal models for GRN deficiency, the portfolio of GRN functions has recently been extended to include functions in important biological processes like energy and protein homeostasis, inflammation as well as neuronal survival, neurite outgrowth, and branching. Furthermore, GRN-deficient animal models have been established and they are believed to be promising disease models as they show accelerated aging and recapitulate at least some neuropathological features of FTD. In this review, we summarize the current knowledge on the molecular mechanisms leading to GRN deficiency and the lessons we learned from the established cellular and animal models. Furthermore, we discuss how these insights might help in developing therapeutic strategies for GRN-associated FTD.     

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ¹. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture ^2-5, in excised ⁶ and living animal tissues ^2,5, and in human tissues in vivo^2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine ^2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) ⁵, and fluoroscein ^6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.     

Interpolating between Cellular Biophysics and Computation in Single Neurons

What types of computations are performed on synaptic inputs within the dendritic trees of single neurons? In this issue of Neuron, present a systematic method to reduce complex, biophysically realistic neuron models to more tractable, simplified two-layered neural networks that could shed some light on this issue.     

Abscission: ethylene and light control

The role of ethylene in light control of leaf abscission im mung bean, Vigna radiata (L.) Wilczek cv Jumbo, cuttings was examined. While red light inhibits and far-red light promotes loss of break strength in abscission zones as compared with dark controls, changes in the rate of abscission could not be associated with changes in the rate of ethylene production. Reducing ethylene synthesis in tissue with aminoethoxyvinylglycine did not alter the effects of red or far-red light on abscission. Far-red light appeared to increase and red light appeared to decrease tissue sensitivity to ethylene.     

Abscission: movement and conjugation of auxin

A 1-hour application of indole-3-acetic acid to bean (Phaseolus vulgaris L. cv. Red Kidney) explants inhibited abscission for an 8-hour aging period. Use of indole-3-acetic acid-¹⁴C showed that the applied indole-3-acetic acid was conjugated within explant tissue and that this conjugation mechanism accounts for loss of effectiveness of indole-3-acetic acid in inhibiting abscission after 8 hours. Reapplication of indole-3-acetic acid to an explant at a later time, before the induced aging requirement was completed reinhibited abscission. 2,4-Dichlorophenoxyacetic acid, which is not destroyed or conjugated by this system, did not lose its ability to inhibit abscission. It was concluded that indole-3-acetic acid destruction is one of the processes involved in the aging stage of abscission in explants.     

Design of a Novel Equi-Biaxial Stretcher for Live Cellular and Subcellular Imaging

Cells in the body experience various mechanical stimuli that are often essential to proper cell function. In order to study the effects of mechanical stretch on cell function, several devices have been built to deliver cyclic stretch to cells; however, they are generally not practical for live cell imaging. We introduce a novel device that allows for live cell imaging, using either an upright or inverted microscope, during the delivery of cyclic stretch, which can vary in amplitude and frequency. The device delivers equi-biaxial strain to cells seeded on an elastic membrane via indentation of the membrane. Membrane area strain was calibrated to indenter depth and the device showed repeatable and accurate delivery of strain at the scale of individual cells. At the whole cell level, changes in intracellular calcium were measured at different membrane area strains, and showed an amplitude-dependent response. At the subcellular level, the mitochondrial network was imaged at increasing membrane area strains to demonstrate that stretch can lead to mitochondrial fission in lung fibroblasts. The device is a useful tool for studying transient as well as long-term mechanotransduction as it allows for simultaneous stretching and imaging of live cells in the presence of various chemical stimuli.     

Kinases and Pseudokinases: Lessons from RAF

Protein kinases are thought to mediate their biological effects through their catalytic activity. The large number of pseudokinases in the kinome and an increasing appreciation that they have critical roles in signaling pathways, however, suggest that catalyzing protein phosphorylation may not be the only function of protein kinases. Using the principle of hydrophobic spine assembly, we interpret how kinases are capable of performing a dual function in signaling. Its first role is that of a signaling enzyme (classical kinases; canonical), while its second role is that of an allosteric activator of other kinases or as a scaffold protein for signaling in a manner that is independent of phosphoryl transfer (classical pseudokinases; noncanonical). As the hydrophobic spines are a conserved feature of the kinase domain itself, all kinases carry an inherent potential to play both roles in signaling. This review focuses on the recent lessons from the RAF kinases that effectively toggle between these roles and can be “frozen” by introducing mutations at their hydrophobic spines.     

Abscission: Response to Red and Far-Red Light

Craker, L. E., Zhao, S. Y. and Decoteau, D. R. 1987. Abscission:response to red and far-red light.—J. exp. Bot. 38: 883–888. The dose-response and time relationship of red and far-red lightin the inhibition and promotion, respectively, of dark-inducedleaf abscission was quantified using cuttings of coleus (ColeusBlumei Benth.). A continuous photon flux of approximately 15nM m^–2 s^–1 of red light was sufficient to preventleaf abscission. Abscission was promoted by exposure to a photonflux of approximately 10 nM m^–2 s^–1 of far-red lightThe inhibition of abscission by red light could be reversedby treatment with far-red and the promotion of abscission byfar-red light could be reversed by treatment with red lightThe data were consistent with a phytochrome receptor systemlocated in the leaves that controlled the presence of an abscission-inhibitingsubstance in the abscission zones. Key words: Abscission, Coleus Blumei, far-red light red light     

Abscission in Coleus: Light and Phytohormone Control

Light control of leaf abscission in Coleus (Coleus blumei Benthcv. Ball 2719 Red) appears to be regulated by the quantity ofendogenous auxin transported from the leaf blade to the abscissionzone. Gas chromatographic—mass spectrophotometric analysisindicated that diffusate collected from leaf tissue treatedwith red light contained significantly higher levels of auxinthan dark and far-red light-treated leaf tissue. In addition,diffusate from red light-treated tissue inhibited abscissionof leafless petioles while diffusate from far-red light-treatedtissue promoted abcission when compared with diffusate fromdark-treated tissue. The effect of red light on abscission couldbe mimicked by IAA, but not by other phytohormones. An auxintransport inhibitor, 2, 3, 5-triiodobenzoic acid (TIBA), appliedeither as a lanolin ring around the petiole or vacuum infiltratedinto tissue, could completely eliminate any red light effecton abscission. The data are consistent with a phytochrome-mediatedlight regulation of endogenous auxin level in the leaf whichthen controls abscission. Key words: Abscission, Coleus, IAA, plant hormones, red (far-red) light, TIBA     

Lessons from yeast for clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) is the major pathway for internalization of membrane proteins from the cell surface. Half a century of studies have uncovered tremendous insights into how a clathrin-coated vesicle is formed. More recently, the advent of live-cell imaging has provided a dynamic view of this process. As CME is highly conserved from yeast to humans, budding yeast provides an evolutionary template for this process and has been a valuable system for dissecting the underlying molecular mechanisms. In this review we trace the formation of a clathrin-coated vesicle from initiation to uncoating, focusing on key findings from the yeast system.     

Systematic Computation of Nonlinear Cellular and Molecular Dynamics with Low-Power CytoMimetic Circuits: A Simulation Study

This paper presents a novel method for the systematic implementation of low-power microelectronic circuits aimed at computing nonlinear cellular and molecular dynamics. The method proposed is based on the Nonlinear Bernoulli Cell Formalism (NBCF), an advanced mathematical framework stemming from the Bernoulli Cell Formalism (BCF) originally exploited for the modular synthesis and analysis of linear, time-invariant, high dynamic range, logarithmic filters. Our approach identifies and exploits the striking similarities existing between the NBCF and coupled nonlinear ordinary differential equations (ODEs) typically appearing in models of naturally encountered biochemical systems. The resulting continuous-time, continuous-value, low-power CytoMimetic electronic circuits succeed in simulating fast and with good accuracy cellular and molecular dynamics. The application of the method is illustrated by synthesising for the first time microelectronic CytoMimetic topologies which simulate successfully: 1) a nonlinear intracellular calcium oscillations model for several Hill coefficient values and 2) a gene-protein regulatory system model. The dynamic behaviours generated by the proposed CytoMimetic circuits are compared and found to be in very good agreement with their biological counterparts. The circuits exploit the exponential law codifying the low-power subthreshold operation regime and have been simulated with realistic parameters from a commercially available CMOS process. They occupy an area of a fraction of a square-millimetre, while consuming between 1 and 12 microwatts of power. Simulations of fabrication-related variability results are also presented.     

Lessons from the past: forests and biodiversity

The biodiversity of forested regions today is the result of complex historical interactions among physical, biological, and social forces over time, often heavily influenced by cycles of various sorts. Fire, agriculture technology, and trade have been particularly powerful human influences on forests. Virtually all of our planet's forests have been affected by the cultural patterns of human use, and the resulting landscape is an ever-changing mosaic of unmanaged and managed patches of habitat, which vary in size, shape, and arrangement. Because chance factors, human influence and small climatic variation can cause very substantial changes in vegetation, the biodiversity for any given landscape will vary substantially over any significant time period- and no one variant is necessarily more natural than the others. This implies that biodiversity conservation efforts may need to give greater attention to ecosystem processes than to ecosystem products. A review of historical evidence shows that past civilizations have tended to over-exploit their forests, and that such abuse of important resources has been a significant factor in the decline of the over-exploiting society. It appears that the best way to maintain biodiversity in forest ecosystems in the late 20th Century is through a combination of strictly protected areas (carefully selected on the basis of clearly defined criteria), multiple-use areas managed by local people, natural forests extensively managed for sustainable yield of logs and other products and services, and forest plantations intensively managed for the wood products needed by society. This diversity of approaches and uses will provide humanity with the widest range of options, the greatest diversity of opportunities, for adapting to the cyclical changes which are certain to continue.     

Opportunities for yeast metabolic engineering: Lessons from synthetic biology

Constant progress in genetic engineering has given rise to a number of promising areas of research that facilitated the expansion of industrial biotechnology. The field of metabolic engineering, which utilizes genetic tools to manipulate microbial metabolism to enhance the production of compounds of interest, has had a particularly strong impact by providing new platforms for chemical production. Recent developments in synthetic biology promise to expand the metabolic engineering toolbox further by creating novel biological components for pathway design. The present review addresses some of the recent advances in synthetic biology and how these have the potential to affect metabolic engineering in the yeast Saccharomyces cerevisiae. While S. cerevisiae for years has been a robust industrial organism and the target of multiple metabolic engineering trials, its potential for synthetic biology has remained relatively unexplored and further research in this field could strongly contribute to industrial biotechnology. This review also addresses are general considerations for pathway design, ranging from individual components to regulatory systems, overall pathway considerations and whole-organism engineering, with an emphasis on potential contributions of synthetic biology to these areas. Some examples of applications for yeast synthetic biology and metabolic engineering are also discussed.