Temperature Imaging using Quadriwave Shearing Interferometry. Applications in Thermoplasmonics

International audienceThe use of illuminated gold nanoparticles as ideal nanosources of heat is the basis of numerous research activities and applications in physics, chemistry, biology and medicine. This field defines the area recently named Thermoplasmonics [1]. In most of the activities related to Thermoplasmonics, probing the temperature at the vicinity of the metal nanoparticles is not an easy task. In this context, we recently developed a novel optical microscopy technique, named TIQSI, aimed at mapping the temperature around plasmonic nanoparticles [2]. The approach is based on the measure of the thermal-induced variation of the refractive index surrounding the sources of heat. The TIQSI technique cumulates all the advantages a thermal microscopy technique may require: i) high resolution (diffraction limited), ii) high readout rate (less than one image per second), iii) high temperature sensitivity (<1°C), iv) large accessible temperature range, v) temperature can be measured without fluorescence labelling or any other kind of thermal probe, v) no need to use sophisticated devices such as heterodyne detection, acousto-optic modulator, spectrometer, etc, like previous thermal imaging techniques. In this presentation, we will first introduce the TIQSI technique, its principle and capabilities. We will then present several recent applications made it possible by this new thermal imaging technique. In particular, we shall explain how this technique have been already used to quantitatively measure the absorption cross section of gold nanoparticles [3] and graphene sheets, how it can be used to map the temperature in real time in living cells [4], how it can help to design temperature distributions at will at the microscale using gold nanoparticles [5,7], and how it can be used to investigate thermal-induced phenomena in hydro- dynamics and phase transitions [6]

The CAMKK2-AMPK kinase pathway mediates the synaptotoxic effects of Aβ oligomers through Tau phosphorylation

Amyloid-β 1-42 (Aβ42) oligomers are synaptotoxic for excitatory cortical and hippocampal neurons and might play a role in early stages of Alzheimer's disease (AD) progression. Recent results suggested that Aβ42 oligomers trigger activation of AMP-activated kinase (AMPK), and its activation is increased in the brain of patients with AD. We show that increased intracellular calcium [Ca²⁺](i) induced by NMDA receptor activation or membrane depolarization activates AMPK in a CAMKK2-dependent manner. CAMKK2 or AMPK overactivation is sufficient to induce dendritic spine loss. Conversely, inhibiting their activity protects hippocampal neurons against synaptotoxic effects of Aβ42 oligomers in vitro and against the loss of dendritic spines observed in the human APP(SWE,IND)-expressing transgenic mouse model in vivo. AMPK phosphorylates Tau on KxGS motif S262, and expression of Tau S262A inhibits the synaptotoxic effects of Aβ42 oligomers. Our results identify a CAMKK2-AMPK-Tau pathway as a critical mediator of the synaptotoxic effects of Aβ42 oligomers

Terminal axon branching is regulated by the LKB1-NUAK1 kinase pathway via presynaptic mitochondrial capture

The molecular mechanisms underlying the axon arborization of mammalian neurons are poorly understood but are critical for the establishment of functional neural circuits. We identified a pathway defined by two kinases, LKB1 and NUAK1, required for cortical axon branching in vivo. Conditional deletion of LKB1 after axon specification or knockdown of NUAK1 drastically reduced axon branching in vivo, whereas their overexpression was sufficient to increase axon branching. The LKB1-NUAK1 pathway controls mitochondria immobilization in axons. Using manipulation of Syntaphilin, a protein necessary and sufficient to arrest mitochondrial transport specifically in the axon, we demonstrate that the LKB1-NUAK1 kinase pathway regulates axon branching by promoting mitochondria immobilization. Finally, we show that LKB1 and NUAK1 are necessary and sufficient to immobilize mitochondria specifically at nascent presynaptic sites. Our results unravel a link between presynaptic mitochondrial capture and axon branching

AMP-activated protein kinase mediates mitochondrial fission in response to energy stress

Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA–linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)–activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission

An 850 nm SiGe/Si HPT with a 4.12 GHz maximum optical transition frequency and 0.805A/W responsivity

A 10 × 10 μm2 SiGe heterojunction bipolar photo-transistor (HPT) is fabricated using a commercial technological process of 80 GHz SiGe bipolar transistors (HBT). Its technology and structure are first briefly described. Its optimal opto-microwave dynamic performance is then analyzed versus voltage biasing conditions for opto-microwave continuous wave measurements. The optimal biasing points are then chosen in order to maximize the optical transition frequency (fTopt) and the opto-microwave responsivity of the HPT. An opto-microwave scanning near-field optical microscopy (OM-SNOM) is performed using these optimum bias conditions to localize the region of the SiGe HPT with highest frequency response. The OM-SNOM results are key to extract the optical coupling of the probe to the HPT (of 32.3%) and thus the absolute responsivity of the HPT. The effect of the substrate is also observed as it limits the extraction of the intrinsic HPT performance. A maximum optical transition frequency of 4.12 GHz and an absolute low frequency opto-microwave responsivity of 0.805A/W are extracted at 850 nm

AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology

Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane-localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.</p

Functionalizing avß3- or a5ß1-selective integrin antagonists for surface coating: A new tool to discriminate integrin subtypes in vitro

Thumbnail image of graphical abstract Stuck with the right choice: avß3- or a5ß1-selective integrin ligands were functionalized for surface coating without losing activity and selectivity. The coating of nanostructured gold surfaces with these compounds stimulated subtype-selective cell adhesion of genetically modified avß3- or a5ß1-expressing fibroblasts in vitro.Postprint (published version

Activity-dependent compartmentalization of dendritic mitochondria morphology through local regulation of fusion-fission balance in neurons in vivo

Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly between the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Ca2+ and Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a signaling pathway underlying the subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise and activity-dependent regulation of mitochondria fission/fusion balance.</p

Thermal Imaging of Nanostructures by Quantitative Optical Phase Analysis

International audienceWe introduce an optical microscopy technique aimed at characterizing the heat generation arising from nanostructures, in a comprehensive and quantitative manner. Namely, the technique permits (i) mapping the temperature distribution around the source of heat, (ii) mapping the heat power density delivered by the source, and (iii) retrieving the absolute absorption cross section of light-absorbing structures. The technique is based on the measure of the thermal-induced refractive index variation of the medium surrounding the source of heat. The measurement is achieved using an association of a regular CCD camera along with a modified Hartmann diffraction grating. Such a simple association makes this technique straightforward to implement on any conventional microscope with its native broadband illumination conditions. We illustrate this technique on gold nanoparticles illuminated at their plasmonic resonance. The spatial resolution of this technique is diffraction limited, and temperature variations weaker than 1 K can be detected

Micropatterning thermoplasmonic gold nanoarrays to manipulate cell adhesion

International audienceThe ability to reversibly control the interactions between the extracellular matrix (ECM) and cell surface receptors such as integrins would allow one to investigate reciprocal signaling circuits between cells and their surrounding environment. Engineering microstructured culture substrates functiona- lized with switchable molecules remains the most adopted strategy to manipulate surface adhesive properties, although these systems exhibit limited reversibility and require sophisticated preparation procedures. Here, we report a straightforward protocol to fabricate biofunctionalized micropatterned gold nanoarrays that favor one-dimensional cell migration and function as plasmonic nanostoves to physically block and orient the formation of new adhesion sites. Being reversible and not restricted spatiotemporally, thermoplasmonic approaches will open new opportunities to further study the complex connections between ECM and cells