Christophe Zimmer

SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope.

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the ‘gene gating’ hypothesis.

Segmenting and tracking fluorescent cells in dynamic 3-D microscopy with coupled active surfaces

Cell migrations and deformations play essential roles in biological processes, such as parasite invasion, immune response, embryonic development, and cancer. We describe a fully automatic segmentation and tracking method designed to enable quantitative analyses of cellular shape and motion from dynamic three-dimensional microscopy data. The method uses multiple active surfaces with or without edges, coupled by a penalty for overlaps, and a volume conservation constraint that improves outlining of cell/cell boundaries. Its main advantages are robustness to low signal-to-noise ratios and the ability to handle multiple cells that may touch, divide, enter, or leave the observation volume. We give quantitative validation results based on synthetic images and show two examples of applications to real biological data.

Segmentation and tracking of migrating cells in videomicroscopy with parametric active contours: a tool for cell-based drug testing

This paper presents a segmentation and tracking method for quantitative analysis of cell dynamics from in vitro videomicroscopy data. The method is based on parametric active contours and includes several adaptations that address important difficulties of cellular imaging, particularly the presence of low-contrast boundary deformations known as pseudopods, and the occurence of multiple contacts between cells. First, we use an edge map based on the average intensity dispersion that takes advantage of relative background homogeneity to facilitate the detection of both pseudopods and interfaces between adjacent cells. Second, we introduce a repulsive interaction between contours that allows correct segmentation of objects in contact and overcomes the shortcomings of previously reported techniques to enforce contour separation. Our tracking technique was validated on a realistic data set by comparison with a manually defined ground-truth and was successfully applied to study the motility of amoebae in a biological research project.

Coupled parametric active contours

We propose an extension of parametric active contours designed to track nonoccluding objects transiently touching each other, a task where both parametric and single level set-based methods usually fail. Our technique minimizes a cost functional that depends on all contours simultaneously and includes a penalty for contour overlaps. This scheme allows us to take advantage of known constraints on object topology, namely, that objects cannot merge. The coupled contours preserve the identity of previously isolated objects during and after a contact event, thus allowing segmentation and tracking to proceed as desired.

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.

Entrapment of Intracytosolic Bacteria by Septin Cage-like Structures

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.

Chromosome arm length and nuclear constraints determine the dynamic relationship of yeast subtelomeres

Physical interactions between distinct chromosomal genomic loci are important for genomic functions including recombination and gene expression, but the mechanisms by which these interactions occur remain obscure. Using telomeric association as a model system, we analyzed here the in vivo organization of chromosome ends of haploid yeast cells during interphase. We separately labeled most of the 32 subtelomeres and analyzed their positions both in nuclear space and relative to three representative reference subtelomeres by high-throughput 3D microscopy and image processing. We show that subtelomeres are positioned nonrandomly at the nuclear periphery, depending on the genomic size of their chromosome arm, centromere attachment to the microtubule organizing center (spindle pole body, SPB), and the volume of the nucleolus. The distance of subtelomeres to the SPB increases consistently with chromosome arm length up to ≈300 kb; for larger arms the influence of chromosome arm length is weaker, but the effect of the nucleolar volume is stronger. Distances between pairs of subtelomeres also exhibit arm-length dependence and suggest, together with dynamic tracking experiments, that potential associations between subtelomeres are unexpectedly infrequent and transient. Our results suggest that interactions between subtelomeres are nonspecific and instead governed by physical constraints, including chromosome structure, attachment to the SPB, and nuclear crowding.

Principles of chromosomal organization: lessons from yeast.

The spatial organization of genes and chromosomes plays an important role in the regulation of several DNA processes. However, the principles and forces underlying this nonrandom organization are mostly unknown. Despite its small dimension, and thanks to new imaging and biochemical techniques, studies of the budding yeast nucleus have led to significant insights into chromosome arrangement and dynamics. The dynamic organization of the yeast genome during interphase argues for both the physical properties of the chromatin fiber and specific molecular interactions as drivers of nuclear order.

FISH-quant: automatic counting of transcripts in 3D FISH images

Transcription is inherently stochastic even in clonal cell populations 1. Studies at single-cell-single-molecule level enable a quantitative understanding of the underlying regulatory mechanisms 2,3. A widely used technique is single-molecule RNA fluorescence in-situ hybridization (FISH), in which fluorescent probes target the mRNA of interest and individual molecules appear as bright diffraction-limited spots (Fig. 1a,b) 3. Recent experimental progress makes FISH easy to use 4 , but a dedicated image analysis tool is currently missing. Available methods allow counting of isolated mature mRNAs but cannot reliably quantify the dense mRNA aggregates at transcription sites (TS) in three dimensions (3D), particularly of highly transcribing genes 4. We developed FISH-QUANT to close this gap (Supplementary Note 1)