Daniel W. Gerlich

Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the ∼21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.

Aurora B-Mediated Abscission Checkpoint Protects against Tetraploidization

Genomic abnormalities are often seen in tumor cells, and tetraploidization, which results from failures during cytokinesis, is presumed to be an early step in cancer formation. Here, we report a cell division control mechanism that prevents tetraploidization in human cells with perturbed chromosome segregation. First, we found that Aurora B inactivation promotes completion of cytokinesis by abscission. Chromosome bridges sustained Aurora B activity to posttelophase stages and thereby delayed abscission at stabilized intercellular canals. This was essential to suppress tetraploidization by furrow regression in a pathway further involving the phosphorylation of mitotic kinesin-like protein 1 (Mklp1). We propose that Aurora B is part of a sensor that responds to unsegregated chromatin at the cleavage site. Our study provides evidence that in human cells abscission is coordinated with the completion of chromosome segregation to protect against tetraploidization by furrow regression.

Nuclear Envelope Breakdown Proceeds by Microtubule-Induced Tearing of the Lamina

The mechanism of nuclear envelope breakdown (NEBD) was investigated in live cells. Early spindle microtubules caused folds and invaginations in the NE up to one hour prior to NEBD, creating mechanical tension in the nuclear lamina. The first gap in the NE appeared before lamin B depolymerization, at the site of maximal tension, by a tearing mechanism. Gap formation relaxed this tension and dramatically accelerated the rate of chromosome condensation. The hole produced in the NE then rapidly expanded over the nuclear surface. NE fragments remaining on chromosomes were removed toward the centrosomes in a microtubule-dependent manner, suggesting a mechanism mediated by a minus-end-directed motor.

Roles of Polo-like Kinase 1 in the Assembly of Functional Mitotic Spindles

BACKGROUND The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. RESULTS We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. CONCLUSIONS Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.

Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments.

The process by which animal cells are physically separated after cell division is dissected in molecular detail. After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The endosomal sorting complex required for transport (ESCRT)–III co-localized with constriction zones and was required for assembly of 17-nanometer-diameter filaments. Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III–mediated fission of large membrane structures.

Global Chromosome Positions Are Transmitted through Mitosis in Mammalian Cells

We investigated positioning of chromosomes during the cell cycle in live mammalian cells with a combined experimental and computational approach. By non-invasive labeling of chromosome subsets and tracking by 4D imaging, we could show that no global rearrangements occurred in interphase. Using the same assay, we also observed a striking order of chromosomes throughout mitosis. By contrast, our computer simulation based on stochastic movements of individual chromosomes predicted randomization of chromosome order in mitosis. In vivo, a quantitative assay for single chromosome positioning during mitosis revealed strong similarities between daughter and mother cells. These results demonstrate that global chromosome positions are heritable through the cell cycle in mammalian cells. Based on tracking of labeled chromosomes and centromeres during chromosome segregation and experimental perturbations of chromosomal order, we propose that chromosome specific timing of sister chromatid separation transmits chromosomal positions from one cell generation to the next.

Fluorogenic probes for live-cell imaging of the cytoskeleton

We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.

Distinct functions of condensin I and II in mitotic chromosome assembly

Condensin is a protein complex associated with mitotic chromosomes that has been implicated in chromosome condensation. In vertebrates, two types of condensin complexes have recently been identified, called condensin I and II. Here, we show that in mammalian cells condensin II associates with chromatin in prophase, in contrast to condensin I which is cytoplasmic and can thus interact with chromosomes only after nuclear envelope breakdown. RNA interference experiments in conjunction with imaging of live and fixed cells revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage. By contrast, condensin I is required for the complete dissociation of cohesin from chromosome arms, for chromosome shortening and for normal timing of progression through prometaphase and metaphase, whereas normal condensin II levels are dispensable for these processes. After depletion of both condensin complexes, the onset of chromosome condensation is delayed until the end of prophase, but is then initiated rapidly before nuclear envelope breakdown. These results reveal that condensin II and I associate with chromosomes sequentially and have distinct functions in mitotic chromosome assembly.

Live-Cell Imaging Reveals a Stable Cohesin-Chromatin Interaction after but Not before DNA Replication

Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation. The lack of obvious cohesin-targeting-specific sequences on DNA, as well as cohesins molecular arrangement as a large ring, has led to the working hypothesis that cohesin acts as a direct topological linker. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging–based screening with assays that directly score cellular dynamics.