Jesse C. Gatlin

Changes in cytoplasmic volume are sufficient to drive spindle scaling.

Scaling Spindle Size The difficulty of modulating cell size in vivo has made it hard to test hypotheses for organelle size scaling during development. To this end, Hazel et al. (p. 853) and Good et al. (p. 856) developed microfluidic systems in which cytoplasmic extracts are encapsulated in compartments with definable size. The size of mitotic spindles assembled within cell-free extracts scaled with the volume of the compartment within which the spindle assembled. The findings suggest that the diminished availability of cytoplasmic components, notably tubulin, concomitant with cell size reduction, prescribes a smaller spindle size. Microfluidic techniques reveal how mitotic spindle size is regulated by titratable cytosolic factors. The mitotic spindle must function in cell types that vary greatly in size, and its dimensions scale with the rapid, reductive cell divisions that accompany early stages of development. The mechanism responsible for this scaling is unclear, because uncoupling cell size from a developmental or cellular context has proven experimentally challenging. We combined microfluidic technology with Xenopus egg extracts to characterize spindle assembly within discrete, geometrically defined volumes of cytoplasm. Reductions in cytoplasmic volume, rather than developmental cues or changes in cell shape, were sufficient to recapitulate spindle scaling observed in Xenopus embryos. Thus, mechanisms extrinsic to the spindle, specifically a limiting pool of cytoplasmic component(s), play a major role in determining spindle size.

Nanoparticle Targeting and Cholesterol Flux Through Scavenger Receptor Type B-1 Inhibits Cellular Exosome Uptake

Exosomes are nanoscale vesicles that mediate intercellular communication. Cellular exosome uptake mechanisms are not well defined partly due to the lack of specific inhibitors of this complex cellular process. Exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts, and can be blocked by non-specific depletion of plasma membrane cholesterol. Scavenger receptor type B-1 (SR-B1), found in lipid rafts, is a receptor for cholesterol-rich high-density lipoproteins (HDL). We hypothesized that a synthetic nanoparticle mimic of HDL (HDL NP) that binds SR-B1 and removes cholesterol through this receptor would inhibit cellular exosome uptake. In cell models, our data show that HDL NPs bind SR-B1, activate cholesterol efflux, and attenuate the influx of esterified cholesterol. As a result, HDL NP treatment results in decreased dynamics and clustering of SR-B1 contained in lipid rafts and potently inhibits cellular exosome uptake. Thus, SR-B1 and targeted HDL NPs provide a fundamental advance in studying cholesterol-dependent cellular uptake mechanisms.

Spatially segregated transcription and translation in cells of the endomembrane-containing bacterium Gemmata obscuriglobus

Significance Eukaryotic (plant and animal) cells possess a nuclear membrane that separates the two stages of gene expression (transcription and translation), whereas prokaryotic (bacteria and archaea) cells lack the nuclear membrane barrier to colocated transcription and translation. However, cells of the bacterium Gemmata obscuriglobus possess extensive intracellular membranes, resulting in superficially eukaryote-like cellular complexity. We have found that a substantial amount of G. obscuriglobus translation is uncoupled from transcription, broadening our understanding of the spatial organization of bacterial gene expression, which currently is based entirely on a handful of model species. This broader understanding provides a useful background for consideration of the evolutionary development of eukaryotic cellular complexity and how it led to decoupled processes of gene expression in eukaryotes. The dogma of coupled transcription and translation in bacteria has been challenged by recent reports of spatial segregation of these processes within the relatively simple cellular organization of the model organisms Escherichia coli and Bacillus subtilis. The bacterial species Gemmata obscuriglobus possesses an extensive endomembrane system. The membranes generate a very convoluted intracellular architecture in which some of the cell’s ribosomes appear to have less direct access to the cell’s nucleoid(s) than others. This observation prompted us to test the hypothesis that a substantial proportion of G. obscuriglobus translation may be spatially segregated from transcription. Using immunofluorescence and immunoelectron microscopy, we showed that translating ribosomes are localized throughout the cell, with a quantitatively greater proportion found in regions distal to nucleoid(s). Our results extend information about the phylogenetic and morphological diversity of bacteria in which the spatial organization of transcription and translation has been studied. These findings also suggest that endomembranes may provide an obstacle to colocated transcription and translation, a role for endomembranes that has not been reported previously for a prokaryotic organism. Our studies of G. obscuriglobus may provide a useful background for consideration of the evolutionary development of eukaryotic cellular complexity and how it led to decoupled processes of gene expression in eukaryotes.

Isolation and Demembranation of Xenopus Sperm Nuclei

The inherent experimental advantages of intact amphibian eggs have been exploited for several decades to advance our understanding of fundamental developmental processes and the cell cycle. Characterization of these processes at the molecular level has been greatly advanced by the use of cell-free extracts, which permit the development of biochemically tractable approaches. Demembranated Xenopus laevis sperm nuclei have been used with cell-free extracts to recapitulate cell cycle progression and to control the cell cycle state of the egg extract. This system has become an invaluable and widely used tool for studies of cell cycle regulation and many downstream events. Here, we describe a protocol, derived in part from other published protocols and modified over time, for the preparation of Xenopus sperm nuclei that can be used in a variety of in vitro assays.

Centrosomal clustering contributes to chromosomal instability and cancer

Cells assemble mitotic spindles during each round of division to insure accurate segregation of their duplicated genome. In animal cells, stereotypical spindles have two poles, each containing one centrosome, from which microtubules are nucleated. By contrast, many cancer cells often contain more than two centrosomes and form transient multipolar spindle structures with more than two poles. In order to divide and produce viable progeny, the multipolar spindle intermediate must be reshaped into a pseudo-bipolar structure via a process called centrosomal clustering. Pseudo-bipolar spindles appear to function normally during mitosis, but they occasionally give rise to aneuploid and transformed daughter cells. Agents that inhibit centrosomal clustering might therefore work as a potential cancer therapy, specifically targeting mitosis in supernumerary centrosome-containing cells.

Tau-based fluorescent protein fusions to visualize microtubules: MOONEY et al.

The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin‐ and microtubule (MT)‐dependent phenomena such as cell motility, cell division, and mitosis. Here, we describe a novel set of fluorescent protein (FP) fusions designed specifically to visualize MTs in living systems using fluorescence microscopy. Each fusion contains a FP module linked in frame to a modified phospho‐deficient version of the MT‐binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the MT‐binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, MT dynamics were not grossly perturbed by the presence of Tau‐based FP fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau‐based FP fusions as minimally perturbing tools to accurately visualize MTs in living systems.

Nucleo-cytoplasmic trafficking regulates nuclear surface area during nuclear organogenesis

Throughout development, nuclei must be assembled following every cell division to establish a functional organelle from compact, mitotic chromatin. During nuclear organogenesis, chromatin expands to establish a nucleus of a given size seperate from the cytoplasm. Determining how nuclear organogenesis is regulated is particularly significant in the context of certain cancers in which scaling relationships between cell and nuclear sizes are not maintained. Controlling cell size in vitro using a microfluidics approach, we determined that neither nuclear volume nor surface area scale directly with cell size. Looking to explain differential nuclear scaling relationships, we developed a simple mechano-chemical mathematical model. In simulating biological perturbations in silico, our model predicted crucial roles for nucleo-cytoplasmic trafficking in regulating nuclear expansion and in restricting the recruitment of a potential nuclear surface area factor. In mammalian tissue culture, inhibiting nuclear export increased nuclear expansion rates and reduced the amount of nuclear lamin, a candidate surface area factor, being recruited to assembling nuclei, supporting our model’s predictions. Targeting the principal nuclear export component in the Drosophila syncytial embryo, Embargoed, we show that nuclear expansion rates are also increased in this developmental context, consistent with our model. Using the MS2-reporter system in fly embryos, we demonstrate a role for nuclear export in regulating transcription activation timing and dynamics, suggesting that regulating nuclear assembly is crucial for downstream nuclear function. Taken together, we propose a simple model through which nuclear organogenesis is achieved and demonstrate a role for nuclear export in regulating nuclear assembly.

Microfluidic Encapsulation of Demembranated Sperm Nuclei in Xenopus Egg Extracts

The cell-free nature of Xenopus egg extract makes it a uniquely tractable experimental model system. The extract, effectively unconfined cytoplasm, allows the direct and relatively straight-forward addition of purified proteins and other reagents, a characteristic that renders the system amenable to many biochemical and cell biological manipulations. Accessibility to the system also facilitates the direct physical manipulation and probing of biological structures, in turn enabling mechanical properties of intracellular assemblies and organelles, such as the mitotic spindle and nucleus, to be measured. Recently, multiphase microfluidics have been combined with Xenopus egg extracts to encapsulate discrete cytoplasmic volumes. Described here is a protocol detailing the use of multiphase microfluidic devices to encapsulate sperm nuclei within extract droplets of defined size and shape. This protocol can also be applied more generally to encapsulation of microbeads and other particles.

Abstract 3673: High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes

Exosomes play a crucial role in the progression of cancer through the transport of a variety molecular cargo, including proteins, lipids, and nucleic acids, to and from cells as a means of intercellular communication. Unraveling mechanisms of exosome-cell interactions may open avenues for studying cellular communication and lead to new therapies. Cellular exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts. Non-specific depletion of lipid raft cholesterol reduces cellular exosome uptake; however, to our knowledge, no targeted mechanism of inhibiting cellular exosome uptake has been reported. Scavenger receptor type B-1 (SR-B1) localizes to lipid rafts, and is a high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL). SR-B1 is an intriguing therapeutic target because it is upregulated in many different cancers due to the high need for cholesterol of rapidly dividing cancer cells. Therefore, we hypothesized that specific targeting of SR-B1 and modulation of cholesterol flux through this receptor with biomimetic HDL-like nanoparticles (HDL NPs) would disrupt cellular exosome uptake. As a model, we explored exosomes derived from melanoma cells as they have been shown to promote angiogenesis and immunosuppression both crucial events in melanoma progression. Melanoma exosomes have also been shown to actively prepare metastatic sites, creating a suitable microenvironment allowing for the development of metastasis. Because of this, targeting exosomes and intercellular signaling could be beneficial for the treatment of metastatic melanoma. Using a variety of techniques including confocal microscopy, flow cytometry and automated image analysis, data demonstrate that HDL NPs specifically target SR-B1 in lipid rafts in melanoma cells and modulate cholesterol flux through this receptor. This leads to a clustering of SR-B1 at the cell membrane and potent inhibition of the cellular uptake of melanoma cell-derived exosomes. Citation Format: Michael P. Plebanek, Alexandre Matov, Kannan Mautharasan, Jesse Gatlin, C. Shad Thaxton. High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2015-3673

Meeting report: mitosis and nuclear structure.

The Company of Biologists Workshop entitled ‘Mitosis and Nuclear Structure’ was held at Wiston House, West Sussex in June 2013. It provided a unique and timely opportunity for leading experts from different fields to discuss not only their own work but also its broader context. Here we present the proceedings of this meeting and several major themes that emerged from the crosstalk between the two, as it turns out, not so disparate fields of mitosis and nuclear structure. Co-chaired by Katherine Wilson (Johns Hopkins School of Medicine, Baltimore, MD), Timothy Mitchison (Harvard University, Cambridge, MA) and Michael Rout (Rockefeller University, New York, NY), this workshop brought together a small group of scientists from a range of disciplines to discuss recent advances and connections between the areas of mitosis and nuclear structure research. Several early-career researchers (students, postdoctoral researchers, junior faculty) participated along with 20 senior scientists, including the venerable and affable Nobel Laureate Tim Hunt. Participants were encouraged to embrace unconventional thinking in the ‘scientific sandbox’ created by this unusual combination of researchers in the inspiring, isolated setting of the 16th-century Wiston House.