Assessment of ORDYL SY 355 dry film for RF MEMS 0-level packaging

RF MEMS must be protected by a suitable package. A simple and cheap solution is to use quartz caps bonded by a polymer. This work reports on the use of ORDYL SY 355, a photosensitive dry film. The caps fabrication and bonding process were developed and tests were performed to define performance. Shear tests demonstrated good adhesion to the substrate and water immersion the sealing capability. Caps bonded on CPW and microstrip lines demonstrated negligible or very low impact on the RF performance in the 0-30GHz tested frequency band. Preliminary tests on capped RF MEMS switches indicated good performance of both capacitive and ohmic contact switches. Yield of ohmic switches resulted more sensitive to process conditions requiring a more accurate control

Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer's disease and regulates axonal transport and processing of the amyloid precursor protein

Damage to axonal transport is an early pathogenic event in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo since disruption to APP transport promotes amyloidogenic processing of APP. Moreover, altered APP processing itself disrupts axonal transport. The mechanisms that regulate axonal transport of APP are therefore directly relevant to Alzheimer's disease pathogenesis. APP is transported anterogradely through axons on kinesin-1 motors and one route for this transport involves calsyntenin-1, a type-1 membrane spanning protein that acts as a direct ligand for kinesin-1 light chains (KLCs). Thus, loss of calsyntenin-1 disrupts APP axonal transport and promotes amyloidogenic processing of APP. Phosphorylation of KLC1 on serine-460 has been shown to reduce anterograde axonal transport of calsyntenin-1 by inhibiting the KLC1-calsyntenin-1 interaction. Here we demonstrate that in Alzheimer's disease frontal cortex, KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased; these changes occur relatively early in the disease process. We also show that a KLC1 serine-460 phosphomimetic mutant inhibits axonal transport of APP in both mammalian neurons in culture and in Drosophila neurons in vivo. Finally, we demonstrate that expression of the KLC1 serine-460 phosphomimetic mutant promotes amyloidogenic processing of APP. Together, these results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer's disease

Clonal imaging of mitochondria in the dissected fly wing

Mitochondria are essential for long-term neuronal function and survival. They are maintained in neurons, including long axonal stretches, through dynamic processes such as fission, fusion, biogenesis and mitophagy. Here we describe a protocol for the in-depth morphological analysis of individual mitochondria in axons in vivo. Much of the mitochondrial analysis of axons is currently achieved in vitro where neurons are in a developmental state. Therefore, understanding the axonal mitochondrial network during aging in fully differentiated neurons and the long-term consequence of gene knockout is often missed. By using a clonal system paired with fluorescent genetically encoded markers in the Drosophila wing, we can visualize individual neurons (out of the whole bundle) including their long axons and the mitochondria that they contain using confocal imaging. The clonal system also allows visualization of neurons with genetic perturbations that would otherwise be lethal if present in the whole organism, allowing investigators to bypass lethality. This protocol can further be adapted to measure the physiological and biochemical state of the mitochondrion. Mitochondrial morphology and health in axons is tightly linked to aging, axon injury, and neurodegeneration; therefore, this method can be used to investigate the mitochondrial dysfunction associated with novel genes or those linked to neurodegenerative disease and axonopath

Optogenetic cleavage of the Miro GTPase reveals the direct consequences of real-time loss of function in Drosophila.

Miro GTPases control mitochondrial morphology, calcium homeostasis, and regulate mitochondrial distribution by mediating their attachment to the kinesin and dynein motor complex. It is not clear, however, how Miro proteins spatially and temporally integrate their function as acute disruption of protein function has not been performed. To address this issue, we have developed an optogenetic loss of function "Split-Miro" allele for precise control of Miro-dependent mitochondrial functions in Drosophila. Rapid optogenetic cleavage of Split-Miro leads to a striking rearrangement of the mitochondrial network, which is mediated by mitochondrial interaction with the microtubules. Unexpectedly, this treatment did not impact the ability of mitochondria to buffer calcium or their association with the endoplasmic reticulum. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows that Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. In adult fly neurons in vivo, Split-Miro photocleavage affects both mitochondrial trafficking and neuronal activity. Furthermore, functional replacement of endogenous Miro with Split-Miro identifies its essential role in the regulation of locomotor activity in adult flies, demonstrating the feasibility of tuning animal behaviour by real-time loss of protein function

Live imaging of mitochondria in the intact fly wing

Detailed mechanisms governing the transport of mitochondria in neurons have recently emerged, although it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. Here, we provide a protocol to study the intracellular dynamics of mitochondria in the wing neurons of adult Drosophila in situ. The mounting and imaging procedures that we describe are suitable for use on most microscopes, and they can be easily implemented in any laboratory. Our noninvasive mounting procedures, combined with the translucency of the wing cuticle in adult animals, makes the wing nervous system accessible to advanced microscopy studies in a physiological environment. Combining the powerful genetics of Drosophila with time-lapse live imaging, users of this protocol will be able to analyze mitochondrial dynamics over time in a subset of sensory neurons in the wing. These cells extend long axons with a stereotypical plus-end-out microtubule orientation that represents a unique model to understand the logic of neuronal cargo transport, including the mitochondria. Finally, the neurons in this tissue respond to mechanical and chemical stimulation of the sensory organs of the wing, opening up the possibility of coupling the study of mitochondrial dynamics with the modulation of neuronal activity in aging Drosophila. We anticipate that the unique characteristics of this in vivo system will contribute to the discovery of novel mechanisms that regulate mitochondrial dynamics within an organismal context with relevant implications for the pathogenesis of age-dependent neurological disorders

Analysis of mitochondrial dynamics in adult drosophila axons

Neuronal survival depends on the generation of ATP from an ever-changing mitochondrial network. This requires a fine balance between the constant degradation of damaged mitochondria, biogenesis of new mitochondria, movement along microtubules, dynamic processes, and adequate functional capacity to meet firing demands. The distribution of mitochondria needs to be tightly controlled throughout the entire neuron, including its projections. Axons in particular can be enormous structures compared to the size of the cell soma, and how mitochondria are maintained in these compartments is poorly defined. Mitochondrial dysfunction in neurons is associated with aging and neurodegenerative diseases, with the axon being preferentially vulnerable to destruction. Drosophila offer a unique way to study these organelles in fully differentiated adult neurons in vivo. Here, we briefly review the regulation of neuronal mitochondria in health, aging, and disease and introduce two methodological approaches to study mitochondrial dynamics and transport in axons using the Drosophila wing system

Insight into the regulation of mitochondrial motility in the processes of S2R+ cells by endogenous Miro and Split-Miro.

(A) Representative western blots of Miro from total lysates of control and Miro RNAi-treated S2R+ cells. (B) Duty cycle analysis describes the average time mitochondria spend moving anterogradely, retrogradely, or pausing. For each parameter, all mitochondrial values from each cell were averaged and compared between control and Miro dsRNA condition using a multiple Student t tests. Number of mitochondria analysed are in brackets from 29 (Ctrl dsRNA) and 36 (Miro dsRNA) cells, respectively, from 2 independent experiments. (C) Run velocities of short and long runs in control dsRNA-treated S2R+ cells showing that Miro-dependent long runs are significantly more processive than the short, Miro-independent runs (Fig 2C). Number of runs analysed are in brackets, from 2 independent experiments. Mann–Whitney test. (D) Split-Miro interacts with Milton in S2R+ cells. Cells were transfected with Split-Miro or Control (as shown in E) and the total cell lysates immunoprecipitated using GFP-beads to pull down EGFP-tagged Split-Miro C-terminus. Immunoprecipitates were blotted and probed with anti-GFP antibody (to detect Split-Miro C-terminus), anti-Miro antibody (to detect Split-Miro N-terminus), and an anti-Milton antibody. Inputs are total lysates (25 μg protein). (E) Cartoon showing Split-Miro and Control constructs with the GFP and Miro antibodies used for immunoprecipitation and western blotting in (D). (F) Representative kymographs of mitochondrial transport in the processes of S2R+ cells transfected with mCherry-tagged Zdk1-MiroC (Control), mCherry-Miro (wt-Miro), and mCherry-Split-Miro (Split-Miro). Scale bars: 2 μm (distance) and 5 seconds (time). G) Distribution of mitochondria run lengths in the processes of S2R+ cells, transfected with control, wt-Miro, and Split-Miro, as shown in F. N = number of mitochondrial runs. One-way ANOVA with Tukey’s post hoc test. (H) Duty cycle analysis describing the average time mitochondria spend moving anterogradely, retrogradely, or pausing in control, wt-Miro, and Split-Miro–transfected cells, relative to (F). For each parameter, all mitochondrial values per cell were averaged and compared by one-way ANOVA followed by Tukey’s post hoc test. Number of mitochondria are in brackets from 16 (control), 15 (wt-Miro), and 15 (Split-Miro) cells, respectively, from 3 independent experiments. (I) S2R+ cells transfected with wt-Miro and Split-Miro were imaged for 1 minute with a 561-nm laser, to capture the mCherry signal, followed by 1-minute imaging with 488-nm blue light, to capture the EGFP signal after Split-Miro photocleavage (relative to Fig 2E). Number of mitochondria are in brackets from 11 (wt-Miro) and 17 (Split-Miro) cells, from 3 independent experiments. Data are shown as mean ± SEM. Kolmogorov–Smirnov test showed no statistical difference between groups. (J) Distribution of mitochondrial run lengths in the anterograde direction in the processes of S2R+ cells, during the first and seventh minute of time-lapse imaging with blue light in cells transfected with wt-Miro or Split-Miro. N = number of runs. Mann–Whitney test showed no statistical difference between groups. (K) Run velocities for long processive anterograde and retrograde runs in S2R+ cells transfected with Split-Miro and Miro dsRNA (which targets endogenous Miro) and imaged by time-lapse for 7 minutes under blue light. Circles, number of runs, from 2 independent experiments. Mann–Whitney test shows no difference between first and seventh minutes of imaging, with the velocities remaining high compared to nontransfected condition (e.g., Figs 2H and S2C). This result shows that the velocities of the processive mitochondria, augmented as a consequence of Split-Miro overexpression, remain elevated even after reduction of endogenous Miro, suggesting Miro is not necessary for maintaining mitochondrial velocities. * p p p S1 Data. (TIF)</p