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Using confocal and lattice light sheet microscopy, the authors perform systems-level analysis of the organelle interactome in live cells, allowing them to visualize the frequency and locality of up to five-way interactions between different organelles.

Live-cell imaging of mRNA encoding secretome proteins and translated nascent peptide markers show that secretome translation occurs at endoplasmic reticulum junctions near lysosomes, requires lunapark protein and is modulated by nutrient status.

During mitosis, complementary actin-based mechanisms ensure equal and random distributions of mitochondria among daughter cells following symmetrical cell division.

Endosomes have important roles in processes, including cytokinesis, polarization and migration, in which their function might be distinct from those classically associated with endosomes. We speculate that endosomes function as multifunctional platforms on which unique sets of molecular machines are assembled to suit different cellular roles.

The skin of zebrafish is patterned by alternating blue stripes and yellow interstripes which arises from guanine crystal-containing cells called iridophores that reflect light. Here the authors track iridophores and see that they do not migrate between stripes and interstripes, but instead differentiate and proliferate in place based on their micro-environment.

Many membrane proteins are taken up into the cell by the process of clathrin-mediated endocytosis — the proteins are pinched off in vesicles coated with the protein clathrin. A study using a fusion protein (green fluorescent protein combined with the clathrin light-chain) now indicates that formation of these coated pits may be coupled to events at the membrane skeleton, possibly through scaffold proteins.

Super-resolution microscopy is poised to revolutionize our understanding of the workings of the cell. But the technology still has some limitations, and these must be taken into consideration if widespread application is to yield biological insight.

Focused ion beam scanning electron microscopy (FIB-SEM) combined with deep-learning-based segmentation is used to produce three-dimensional reconstructions of complete cells and tissues, in which up to 35 different organelle classes are annotated.

How PDZD8 acted in Mitochondria-ER contact formation was unclear. Here, the authors show that binding to mitochondrial FKBP8 slows PDZD8’s rapid motion, enhancing contacts and mitochondrial complexity.

Improved photoactivatable red fluorescent proteins are generated by including properties desirable for photoactivated localization microscopy (PALM) as selection criteria. The PAmCherry proteins are superior tags for one- and two-color PALM in fixed cells, among other applications. Also in this issue, McKinney et al. present an improved version of the green-to-red EosFP protein.

Actin condenses at the lamellipodium of migrating cells to form arc-like bundles parallel to the leading edge. During the retraction phase of the edge movement, these arcs are shown to be displaced towards the rear of the lamella, and their movement slows down when they join focal adhesions. Actin arcs thus provide a spatiotemporal connection between the lamellipodium and the lamella.

This study has reconstituted membrane invagination and scission in vitro using purified endosomal sorting complex required for transport (ESCRT)-III subunits and giant unilamellar vesicles. Snf7 is important for membrane scission whereas the ATPase Vps4 is required for recycling ESCRT-III proteins for further rounds of membrane budding.

Rationalized deep learning improves image reconstruction by incorporating prior knowledge of illumination patterns.

High-speed molecular tracking is integrated with three-dimensional electron microscopy to map the diffusion distribution and ultrastructure of endoplasmic reticulum-mitochondria contact sites, revealing the ability of high-speed single-molecule imaging to map contact site interface structures and corresponding diffusion landscapes.

The endoplasmic reticulum proteins CLIMP63, kinectin and p180 bind preferentially to subsets of microtubules with different post-translational modifications, thereby linking the ‘tubulin code’ to the intracellular distribution of membrane organelles.

Open-access 3D images of whole cells and tissues with combined finer resolution and larger sample size are enabled by advances in focused ion beam-scanning electron microscopy.

A general tuning strategy is introduced for improving the utility of rhodamines for biological imaging applications. The strategy yielded bright, versatile and bioavailable far-red and near-infrared ‘Janelia Fluor’ dyes.

Cai et al. show that YAP forms liquid-like condensates in the nucleus that compartmentalize YAP’s DNA binding cofactors and transcription co-activators to induce transcription of YAP-specific proliferation genes.

Fas drives apoptosis and mutations in this receptor can cause autoimmunity through failure of cell death. Here, the authors uselpr/lprmice with palmitoylation-defective mutant Fas to provide evidence that Fas might limit spontaneous autoimmunity through a non-apoptotic mechanism.

Raj and colleagues show that ERX-41 inhibits lipase-independent functions of LIPA and induces ER stress in different tumor types, providing a therapeutic strategy in PDX models of pancreas, ovarian and triple-negative breast cancer.

Cells in many tissues fuse into syncytia acquiring new functions. By investigating whether physical remodelling promotes differentiation, here, the authors show that plasma membrane diminution post-fusion causes transient nutrient stress that inhibits YAP1 activity and may reduce proliferation-promoting transcription.

On amino acid deprivation TFEB translocates from the cytoplasm to the nucleus. Here the authors identify a nuclear export signal in TFEB that is recognized by the exportin CRM1, and show that dual phosphorylation at S142 and S138 by mTOR accelerates export of TFEB.

Sengupta et al. show that protein sorting into the membranes of budding HIV particles is dependent on lipid-based partitioning of proteins, through a mechanism initiated by HIV protein Gag oligomerization at viral assembly sites.

Endoplasmic reticulum exit sites (ERES) are specialized ER subdomains that regulate the export of secreted cargo. This Roadmap explores how ERES integrate biochemical and mechanical signals to coordinate trafficking and proposes a multidisciplinary strategy to investigate their function, including in disease.

Emerging evidence suggests that ESCRT proteins, well characterized in their role in multivesicular body trafficking, contribute to various cellular processes including cytokinesis. Structural and functional analyses indicate that the ESCRT-III protein CHMP1B promotes the midbody localization of spastin, a microtubule-severing protein required for membrane abscission.

Bayesian analysis of fluorophore blinking and bleaching in image data collected from simple xenon arc lamp illumination and high-speed wide-field imaging of standard fluorescent proteins allows localization microscopy in living cells with a 50 nm spatial and a 4 s temporal resolution.

This Perspective presents recent insights into vimentin filament structure and function and discusses emerging findings that point to a role in tumour invasion in mesenchymal cells analogous to embryonic development and wound healing.

A FRET-based approach to monitor the assembly of membrane-associated COPII coat proteins provides new insights into how the COPII–cargo complex is maintained on the membrane during continuous rounds of Sar1p-GTP hydrolysis.

Implementation of pair correlation analysis for photoactivated localization microscopy allows quantitative analysis of protein clustering in the plasma membrane, revealing the degree to which different perturbations alter protein arrangements.

Westlake and colleagues discover that membrane shaping EHD proteins participate in ciliogenesis by taking part in ciliary vesicle formation and transition zone protein recruitment.

Live-cell imaging shows that haematopoietic progenitor cells contact osteoblasts through a specialized membrane domain that is consequently internalized by osteoblasts. This results in downregulation of Smad signalling and the production of chemokines by osteoblasts that stimulate haematopoietic progenitor cell homing to bone marrow.