During mitosis in Saccharomyces cerevisiae, senescence factors such as extrachromosomal ribosomal DNA circles (ERCs) are retained in the mother cell and excluded from the bud/daughter cell. Shcheprova et al. proposed a model suggesting segregation of ERCs through their association with nuclear pore complexes (NPCs) and retention of preexisting NPCs in the mother cell during mitosis. However, this model is inconsistent with previous data and we demonstrate here that NPCs do efficiently migrate from the mother into the bud. Therefore, binding to NPCs does not seem to explain the retention of ERCs in the mother cell.

Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations.

Locomotor systems generate diverse motor patterns to produce the movements underlying behavior, requiring that motor neurons be recruited at various phases of the locomotor cycle. Reciprocal inhibition produces alternating motor patterns; however, the mechanisms that generate other phasic relationships between intrasegmental motor pools are unknown. Here, we investigate one such motor pattern in the Drosophila larva, using a multidisciplinary approach including electrophysiology and ssTEM-based circuit reconstruction. We find that two motor pools that are sequentially recruited during locomotion have identical excitable properties. In contrast, they receive input from divergent premotor circuits. We find that this motor pattern is not orchestrated by differential excitatory input but by a GABAergic interneuron acting as a delay line to the later-recruited motor pool. Our findings show how a motor pattern is generated as a function of the modular organization of locomotor networks through segregation of inhibition, a potentially general mechanism for sequential motor patterns.

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder (MDD). The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist of the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity- based drug-sensing fluorescent reporters (“iDrugSnFRs”) targeted to the plasma membrane (PM), cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also employed chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER, at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥ 18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for > 2.4 h. They inhibit SERT transport-associated currents 6- or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the “therapeutic lag” of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the “antidepressant discontinuation syndrome”.

Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed treatment for individuals experiencing major depressive disorder (MDD). The therapeutic mechanisms that take place before, during, or after SSRIs bind the serotonin transporter (SERT) are poorly understood, partially because no studies exist of the cellular and subcellular pharmacokinetic properties of SSRIs in living cells. We studied escitalopram and fluoxetine using new intensity-based drug-sensing fluorescent reporters ("iDrugSnFRs") targeted to the plasma membrane (PM), cytoplasm, or endoplasmic reticulum (ER) of cultured neurons and mammalian cell lines. We also employed chemical detection of drug within cells and phospholipid membranes. The drugs attain equilibrium in neuronal cytoplasm and ER, at approximately the same concentration as the externally applied solution, with time constants of a few s (escitalopram) or 200-300 s (fluoxetine). Simultaneously, the drugs accumulate within lipid membranes by ≥ 18-fold (escitalopram) or 180-fold (fluoxetine), and possibly by much larger factors. Both drugs leave cytoplasm, lumen, and membranes just as quickly during washout. We synthesized membrane-impermeant quaternary amine derivatives of the two SSRIs. The quaternary derivatives are substantially excluded from membrane, cytoplasm, and ER for > 2.4 h. They inhibit SERT transport-associated currents 6- or 11-fold less potently than the SSRIs (escitalopram or fluoxetine derivative, respectively), providing useful probes for distinguishing compartmentalized SSRI effects. Although our measurements are orders of magnitude faster than the "therapeutic lag" of SSRIs, these data suggest that SSRI-SERT interactions within organelles or membranes may play roles during either the therapeutic effects or the "antidepressant discontinuation syndrome".Selective serotonin reuptake inhibitors stabilize mood in several disorders. In general, these drugs bind to the serotonin (5-hydroxytryptamine) transporter (SERT), which clears serotonin from CNS and peripheral tissues. SERT ligands are effective and relatively safe; primary care practitioners often prescribe them. However, they have several side effects and require 2 to 6 weeks of continuous administration until they act effectively. How they work remains perplexing, contrasting with earlier assumptions that the therapeutic mechanism involves SERT inhibition followed by increased extracellular serotonin levels. This study establishes that two SERT ligands, fluoxetine and escitalopram, enter neurons within minutes, while simultaneously accumulating in many membranes. Such knowledge will motivate future research, hopefully revealing where and how SERT ligands "engage" their therapeutic target(s).

Fluorescence microscopy and fluorescent protein (FP)-tagged GLUT4 molecule have been great tools to characterize GLUT4 localization and dynamics inside the cell. However, it was difficult to distinguish GLUT4 storage vesicles (GSVs) from other intracellular compartments containing GLUT4 in live cells. Here, we describe the use of IRAP-pHluorin and total internal reflection fluorescence (TIRF) microscopy to selectively visualize GSVs and Rab proteins that associate with GSVs. This assay is also valuable to further defining GSV identity by unraveling other GSV-associated proteins.

The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

Commentary: Our goal as tool developers is to invent methods capable of uncovering new biological insights unobtainable by pre-existing technologies. A terrific example is given by this paper, where grad students Derek Greenfield and Ann McEvoy in Jan Liphardt’s group at Berkeley used our PALM to image the size and position distributions of chemotaxis proteins in E. Coli with unprecedented precision and sensitivity. Their analysis revealed that the cluster sizes follow a stretched exponential distribution, and the density of clusters is highest furthest away from the largest (e.g., polar) clusters. Both observations support a model for passive self-assembly rather than active cytoskeletal assembly of the chemotaxis network.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Reconstructing neuronal circuits at the level of synapses is a central problem in neuroscience, and the focus of the nascent field of connectomics. Previously used to reconstruct the C. elegans wiring diagram, serial-section transmission electron microscopy (ssTEM) is a proven technique for the task. However, to reconstruct more complex circuits, ssTEM will require the automation of image processing. We review progress in the processing of electron microscopy images and, in particular, a semi-automated reconstruction pipeline deployed at Janelia. Drosophila circuits underlying identified behaviors are being reconstructed in the pipeline with the goal of generating a complete Drosophila connectome.

We present semiparametric spectral modeling of the complete larval Drosophila mushroom body connectome. Motivated by a thorough exploratory data analysis of the network via Gaussian mixture modeling (GMM) in the adjacency spectral embedding (ASE) representation space, we introduce the latent structure model (LSM) for network modeling and inference. LSM is a generalization of the stochastic block model (SBM) and a special case of the random dot product graph (RDPG) latent position model, and is amenable to semiparametric GMM in the ASE representation space. The resulting connectome code derived via semiparametric GMM composed with ASE captures latent connectome structure and elucidates biologically relevant neuronal properties.