Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells

Gerlich, Daniel W.; Beaudouin, Joël; Gebhard, Matthias; Ellenberg, Jan; Eils, Roland

doi:10.1038/ncb0901-852

Cited by 96 publications

(71 citation statements)

References 26 publications

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“…4D multicolor live cell imaging and image processing Live cell imaging was performed at 37°C maintained by an air stream incubator (ASI 400; Nevtek, Burnsville, VA) in conjunction with an objective heater (Bioptechs, Butler, PA) on a customized LSM 510 confocal microscope fitted with a z-scanning stage, selected photomultiplier tubes (Carl Zeiss), a Kr 413 nm laser (Coherent, Dieburg, Germany) and custom dichroics and emission filters (Chroma, Brattleboro, VT) for fluorescent protein imaging as described elsewhere (Daigle et al, 2001;Gerlich et al, 2001). Mitotic events were monitored with a PlanApochromat 63× N.A.…”

Section: Dna Constructsmentioning

confidence: 99%

LAP2α and BAF transiently localize to telomeres and specific regions on chromatin during nuclear assembly

Dechat

Gajewski

Korbei

et al. 2004

Journal of Cell Science

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Lamina-associated polypeptide (LAP) 2α is a LEM (lamina-associated polypeptide emerin MAN1) family protein associated with nucleoplasmic A-type lamins and chromatin. Using live cell imaging and fluorescence microscopy we demonstrate that LAP2α was mostly cytoplasmic in metaphase and associated with telomeres in anaphase. Telomeric LAP2α clusters grew in size, formed `core' structures on chromatin adjacent to the spindle in telophase, and translocated to the nucleoplasm in G1 phase. A subfraction of lamin C and emerin followed LAP2α to the core region early on, whereas LAP2β, lamin B receptor and lamin B initially bound to more peripheral regions of chromatin, before they spread to core structures with different kinetics. Furthermore, the DNA-crosslinking protein barrier-to-autointegration factor (BAF) bound to LAP2α in vitro and in mitotic extracts, and subfractions of BAF relocalized to core structures with LAP2α. We propose that LAP2α and a subfraction of BAF form defined complexes in chromatin core regions and may be involved in chromatin reorganization during early stages of nuclear assembly.

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Section: Dna Constructsmentioning

confidence: 99%

LAP2α and BAF transiently localize to telomeres and specific regions on chromatin during nuclear assembly

Dechat

Gajewski

Korbei

et al. 2004

Journal of Cell Science

Self Cite

188

206

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“…However, only a few studies of cytoplasmic transport have focused on quantitative biophysical parameters such as effective diffusion constants or transport velocities (4)(5)(6). Most experiments measuring these parameters have focused on examining small injected oligonucleotides or proteins (7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy

Kulkarni

Davis

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Quantitatively understanding how nonviral gene delivery vectors (polyplexes) are transported inside cells is essential before they can be optimized for gene therapy and medical applications. In this study, we used spatio-temporal image correlation spectroscopy (ICS) to follow polymer-nucleic acid particles (polyplexes) of various sizes and analyze their diffusive-like and flow behaviors intracellularly to elucidate the mechanisms responsible for their transport. ICS is a quantitative imaging technique that allows the assessment of particle motion in complex systems, although it has not been widely used to date. We find that the internalized polyplexes are able to use microtubule motors for intracellular trafficking and exhibit different transport behaviors for short (<10 s) versus long (Ϸ60 s) correlation times. This motion can be explained by a memory effect of the microtubule motors. These results reveal that, although microtubule motor biases may be present for short periods of time, resulting in a net directional velocity, the overall long-term motion of the polyplexes is best described as a random walk-like process. These studies suggest that spatio-temporal ICS is a powerful technique for assessing the nature of intracellular motion and provides a quantitative tool to compare the transport of different objects within a living cell.transfection agent ͉ molecular mobility ͉ cytoplasmic crowding ͉ cyclodextrin R ecently, significant progress has been made in developing alternatives to viral-based gene therapy, motivated by safety concerns of viral infection. Understanding such nonviral gene delivery (one type of vector that involves the combination of synthetic polymers and nucleic acids to form particles called polyplexes and is used here) requires knowledge of how they behave within cells, including the mechanisms involved in polyplex trafficking such as endocytosis, cytoplasmic transport, endosomal escape, and nuclear localization (1-3). Recent reports have indicated the presence of significant bottlenecks in the delivery process, especially problems with endosomal escape (4). Measuring these dynamics, especially transport parameters, is important for understanding both how such delivery methods compare with viruses and how to improve their efficiencies.Confocal microscopy allows for good visualization of small quantities of fluorescently labeled species. However, only a few studies of cytoplasmic transport have focused on quantitative biophysical parameters such as effective diffusion constants or transport velocities (4-6). Most experiments measuring these parameters have focused on examining small injected oligonucleotides or proteins (7-11). In contrast, polyplexes that range in size from 75 to 250 nm or larger enter cells by endocytosis and become enclosed in endosomes (12). For transport of large entities (Ͼ30 nm), such as the polyplexes used for gene therapy, issues such as crowding and microtubule transport become critical (13,14).Here, we describe spatio-temporal image correlation spectroscopy (IC...

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“…For instance, cryoelectron tomography has enabled the reconstruction of high-resolution, 3-dimensional models of organelle structure [the mitochondrion (2), the Golgi apparatus (3), and the endoplasmic reticulum (3)] as well as HIV viral entry (4), and the endocytotic machinery (5). Similarly, confocal and ''super-resolution'' fluorescence microscopy have emerged as a powerful tool for the capture of 3-dimensional cellular structure (6)(7)(8).…”

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confidence: 99%

Membrane shape as a reporter for applied forces

Lee

Peterson

Phillips

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Recent advances have enabled 3-dimensional reconstructions of biological structures in vivo, ranging in size and complexity from single proteins to multicellular structures. In particular, tomography and confocal microscopy have been exploited to capture detailed 3-dimensional conformations of membranes in cellular processes ranging from viral budding and organelle maintenance to phagocytosis. Despite the wealth of membrane structures available, there is as yet no generic, quantitative method for their interpretation. We propose that by modeling these observed biomembrane shapes as fluid lipid bilayers in mechanical equilibrium, the externally applied forces as well as the pressure, tension, and spontaneous curvature can be computed directly from the shape alone. To illustrate the potential power of this technique, we apply an axial force with optical tweezers to vesicles and explicitly demonstrate that the applied force is equal to the force computed from the membrane conformation.lipid bilayers ͉ membrane mechanics ͉ optical trapping ͉ membrane tethering

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Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells

Cited by 96 publications

References 26 publications

LAP2α and BAF transiently localize to telomeres and specific regions on chromatin during nuclear assembly

LAP2α and BAF transiently localize to telomeres and specific regions on chromatin during nuclear assembly

Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy

Membrane shape as a reporter for applied forces

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