This paper describes an imaging microscopic technique based on heterodyne digital holography where subwavelength-sized gold colloids can be imaged in cell environments. Surface cellular receptors of 3T3 mouse fibroblasts are labeled with 40 nm gold nanoparticles, and the biological specimen is imaged in a total internal reflection configuration with holographic microscopy. Due to a higher scattering efficiency of the gold nanoparticles versus that of cellular structures, accurate localization of a gold marker is obtained within a 3D mapping of the entire sample’s scattered field, with a lateral precision of 5 nm and 100 nm in the x,y and in the z directions respectively, demonstrating the ability of holographic microscopy to locate nanoparticles in living cell environments.

VAMP7 is involved in autophagy and in exocytosis-mediated neurite growth, two yet unconnected cellular pathways. Here, we find that nutrient restriction and activation of autophagy stimulate axonal growth, while autophagy inhibition leads to loss of neuronal polarity. VAMP7 knockout (KO) neuronal cells show impaired neurite growth, whereas this process is increased in autophagy-null ATG5 KO cells. We find that endoplasmic reticulum (ER)-phagy-related LC3-interacting-region-containing proteins Atlastin 3 and Reticulon 3 (RTN3) are more abundant in autophagy-related protein ATG5 KO and less abundant in VAMP7 KO secretomes. Treatment of neuronal cells with ATG5 or VAMP7 KO conditioned medium does not recapitulate the effect of these KOs on neurite growth. A nanobody directed against VAMP7 inhibits axonal overgrowth induced by nutrient restriction. Furthermore, expression of the inhibitory Longin domain of VAMP7 impairs the subcellular localization of RTN3 in neurons. We propose that VAMP7-dependent secretion of RTN3 regulates neurite growth.

While contraction of sarcomeric actomyosin assemblies is well understood, this is not the case for disordered networks of actin filaments (F-actin) driving diverse essential processes in animal cells. For example, at the onset of meiosis in starfish oocytes a contractile F-actin network forms in the nuclear region transporting embedded chromosomes to the assembling microtubule spindle. Here, we addressed the mechanism driving contraction of this 3D disordered F-actin network by comparing quantitative observations to computational models. We analyzed 3D chromosome trajectories and imaged filament dynamics to monitor network behavior under various physical and chemical perturbations. We found no evidence of myosin activity driving network contractility. Instead, our observations are well explained by models based on a disassembly-driven contractile mechanism. We reconstitute this disassembly-based contractile system in silico revealing a simple architecture that robustly drives chromosome transport to prevent aneuploidy in the large oocyte, a prerequisite for normal embryonic development.