Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, and approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H out of the matrix to maintain mitochondrial membrane potential.

ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the lamprey central canal.

Cerebrospinal fluid-contacting (CSF-c) neurons line the central canal of the spinal cord and a subtype of CSF-c neurons expressing somatostatin, forms a homeostatic pH regulating system. Despite their importance, their intricate spatial organization is poorly understood. The function of another subtype of CSF-c neurons expressing dopamine is also investigated.

RecA finds homologous DNA by reduced dimensionality search.

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs). Initially, the RecBCD complex resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles. Next, the filament locates the homologous repair template on the sister chromosome.

STED and parallelized RESOLFT optical nanoscopy of the tubular endoplasmic reticulum and its mitochondrial contacts in neuronal cells.

The classic view of organelle cell biology is undergoing a constant revision fueled by the new insights unraveled by fluorescence nanoscopy, which enable sensitive, faster and gentler observation of specific proteins in situ. The endoplasmic reticulum (ER) is one of the most challenging structure to capture due the rapid and constant restructuring of fine sheets and tubules across the full 3D cell volume. Here we apply STED and parallelized 2D and 3D RESOLFT live imaging to uncover the tubular ER organization in the fine processes of neuronal cells with focus on mitochondria-ER contacts, which recently gained medical attention due to their role in neurodegeneration.

Volumetric live cell imaging with three-dimensional parallelized RESOLFT microscopy.

Elucidating the volumetric architecture of organelles and molecules inside cells requires microscopy methods with a sufficiently high spatial resolution in all three dimensions. Current methods are limited by insufficient resolving power along the optical axis, long recording times and photobleaching when applied to live cell imaging. Here, we present a 3D, parallelized, reversible, saturable/switchable optical fluorescence transition (3D pRESOLFT) microscope capable of delivering sub-80-nm 3D resolution in whole living cells.

Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers.

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression.

Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo.

RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70-90% for in vivo imaging.

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems.

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45-65 nm within the entire cell volume at low light intensities (W-kW cm). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined to gain fluorescence ON-OFF switching cycles and image contrast.

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols.

Investigating the effect of cell substrate on cancer cell stiffness by optical tweezers.

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17kPa respectively).

Vimentin Levels and Serine 71 Phosphorylation in the Control of Cell-Matrix Adhesions, Migration Speed, and Shape of Transformed Human Fibroblasts.

Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30-50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy.

Effect of neighboring cells on cell stiffness measured by optical tweezers indentation.

We report on the modification of mechanical properties of breast cancer cells when they get in contact with other neighboring cells of the same type. Optical tweezers vertical indentation was employed to investigate cell mechanics in isolated and contact conditions, by setting up stiffness as a marker. Two human breast cancer cell lines with different aggressiveness [MCF-7 (luminal breast cancer) and MDA-MB-231 (basal-like breast cancer)] and one normal immortalized breast cell line HBL-100 (normal and myoepithelial) were selected.

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells.

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.

Time-resolved FT-IR microspectroscopy of protein aggregation induced by heat-shock in live cells.

Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress.