Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity, and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signaling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a \~{}40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signaling layer containing integrin cytoplasmic tails, focal adhesion kinase, and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin’s polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

Luminescent centers with sharp (<0.07 millielectron volt), spectrally distinct emission lines were imaged in a GaAs/AIGaAs quantum well by means of low-temperature near-field scanning optical microscopy. Temperature, magnetic field, and linewidth measurements establish that these centers arise from excitons laterally localized at interface fluctuations. For sufficiently narrow wells, virtually all emission originates from such centers. Near-field microscopy/spectroscopy provides a means to access energies and homogeneous line widths for the individual eigenstates of these centers, and thus opens a rich area of physics involving quantum resolved systems.

Luminescent centers with sharp (<0.07 millielectron volt), spectrally distinct emission lines were imaged in a GaAs/AIGaAs quantum well by means of low-temperature near-field scanning optical microscopy. Temperature, magnetic field, and linewidth measurements establish that these centers arise from excitons laterally localized at interface fluctuations. For sufficiently narrow wells, virtually all emission originates from such centers. Near-field microscopy/spectroscopy provides a means to access energies and homogeneous line widths for the individual eigenstates of these centers, and thus opens a rich area of physics involving quantum resolved systems.

Commentary: Harald Hess and I joined forces, combining my near-field optical technology with his cryogenic scanned probe microscope to produce the first paper on high resolution spectroscopy beyond the diffraction limit. We discovered that the broad luminescence spectrum traditionally observed from quantum well heterostructures reflects a resolution-limited ensemble average of emission from numerous discrete sites of exciton recombination occurring at atomic-scale corrugations in the confining interfaces. With the combination of high spatial resolution from near-field excitation and high spectral resolution from cryogenic operation, we were able to isolate these emission sites in a multidimensional space of xy position and wavelength, even though their density was too great to isolate them on the basis of spatial resolution alone. This insight was very influential in the genesis of the concept (see above) that would eventually lead to far-field superresolution by PALM.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial β-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at ∼1 μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca channels and ER Ca-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca homeostasis, and local activation of the Ca/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca modulatory machinery, facilitating signal transmission and ryanodine-receptor-dependent Ca release at ER-PM junctions over 20 μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.

Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

Cells display complex intracellular organization by compartmentalization of metabolic processes into organelles, yet the resolution of these structures in the native tissue context and their functional consequences are not well understood. Here we resolved the three-dimensional structural organization of organelles in large (more than 2.8 × 10 µm) volumes of intact liver tissue (15 partial or full hepatocytes per condition) at high resolution (8 nm isotropic pixel size) using enhanced focused ion beam scanning electron microscopy imaging followed by deep-learning-based automated image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese mice and found substantial alterations, particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural reorganization characterized by marked disorganization of stacks of ER sheets and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes by monitoring the effects of experimental recovery of the subcellular organization on cellular and systemic metabolism. We conclude that the hepatic subcellular organization of the ER architecture are highly dynamic, integrated with the metabolic state and critical for adaptive homeostasis and tissue health.

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two inter-related mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1 and CHMP1B modifying LD membrane morphology. Furthermore, M1 Spastin, IST1 and CHMP1B are all required to relieve LDs of lipid peroxidation. The roles of M1 Spastin in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may help explain the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

Primary cilia are sensory organelles present in many cell types, partaking in various signaling processes. Primary cilia of pancreatic beta cells play pivotal roles in paracrine signaling and their dysfunction is linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of beta cell primary cilia by electron and expansion microscopy. These cilia are spatially confined within deep ciliary pockets or narrow spaces between cells, lack motility components and display an unstructured axoneme organization. Furthermore, we observe a plethora of beta cell cilia-cilia and cilia-cell interactions with other islet and non-islet cells. Most remarkably, we have identified and characterized axo-ciliary synapses between beta cell cilia and the cholinergic islet innervation. These findings highlight the beta cell cilia's role in islet connectivity, pointing at their function in integrating islet intrinsic and extrinsic signals and contribute to understanding their significance in health and diabetes.