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The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

Photobleaching limits extended imaging of fluorescent biological samples. We developed DNA-based FluoroCubes that are similar in size to the green fluorescent protein, have single-point attachment to proteins, have a ~54-fold higher photobleaching lifetime and emit ~43-fold more photons than single organic dyes. We demonstrate that DNA FluoroCubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

Cell division is essential to expand, shape, and replenish epithelia. In the adult small intestine, cells from a common progenitor intermix with other lineages, whereas cell progeny in many other epithelia form contiguous patches. The mechanisms that generate these distinct patterns of progeny are poorly understood. Using light sheet and confocal imaging of intestinal organoids, we show that lineages intersperse during cytokinesis, when elongated interphase cells insert between apically displaced daughters. Reducing the cellular aspect ratio to minimize the height difference between interphase and mitotic cells disrupts interspersion, producing contiguous patches. Cellular aspect ratio is similarly a key parameter for division-coupled interspersion in the early mouse embryo, suggesting that this physical mechanism for patterning progeny may pertain to many mammalian epithelia. Our results reveal that the process of cytokinesis in elongated mammalian epithelia allows lineages to intermix and that cellular aspect ratio is a critical modulator of the progeny pattern.

New technologies can make previously invisible phenomena visible. Nowhere is this more obvious than in the field of light microscopy. Beginning with the observation of "animalcules" by Antonie van Leeuwenhoek, when he figured out how to achieve high magnification by shaping lenses, microscopy has advanced to this day by a continued march of discoveries driven by technical innovations. Recent advances in single-molecule-based technologies have achieved unprecedented resolution, and were the basis of the Nobel prize in Chemistry in 2014. In this article, we focus on developments in camera technologies and associated image processing that have been a major driver of technical innovations in light microscopy. We describe five types of developments in camera technology: video-based analog contrast enhancement, charge-coupled devices (CCDs), intensified sensors, electron multiplying gain, and scientific complementary metal-oxide-semiconductor cameras, which, together, have had major impacts in light microscopy.

μManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, μManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced μManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging.

Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.

Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.

Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets (1,2) enclosed in a monolayer of phospholipids and associated proteins (3,4). These dynamic organelles (5) serve as the principal reservoirs for [...]

In vitro motility assay shows that Unc104 kinesin transports membrane cargo mediated by pH domain of the protein, which binds to the liposomes containing phosphatidylinositol(4,5)bisphosphate. Data indicate that Unc104 clustering in phosphatidylinositol(4,5)bisphosphate lipid rafts may activate membrane transport.