Biocompatible Magnetic Conjugated Polymer Nanoparticles for Optical and Lifetime Imaging Applications in the First Biological Window.

Conjugated polymers are organic semiconductors that can be used for fluorescence microscopy of living specimens. Here, we report the encapsulation of the bright-red-emitting conjugated polymer, poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorenylene}-alt-co-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}] (CN-FO-DPD), and superparamagnetic iron oxide nanoparticles (SPIONs) within poly(styrene-co-maleic anhydride) (PSMA) micelles. The resulting particles exhibited an emission peak at 657 nm, a fluorescence quantum yield of 21%, an average diameter of 65 nm, and a ζ potential of -30 mV. They are taken up by cells, and we describe their use in fluorescence microscopy of living Hela cells and zebrafish embryos and their associated cytotoxicity in HEK, HeLa, and HCE cells.

Removal of Stomatin, a Membrane-Associated Cell Division Protein, Results in Specific Cellular Lipid Changes.

Lipids are key constituents of all cells, which express thousands of different lipid species. In most cases, it is not known why cells synthesize such diverse lipidomes, nor what regulates their metabolism. Although it is known that dividing cells specifically regulate their lipid content and that the correct lipid complement is required for successful division, it is unclear how lipids connect with the cell division machinery. Here, we report that the membrane protein stomatin is involved in the cytokinesis step of cell division. Although it is not a lipid biosynthetic enzyme, depletion of stomatin causes cells to change their lipidomes. These changes include specific lipid species, like ether lipids, and lipid families like phosphatidylcholines. Addition of exogenous phosphatidylcholines rescues stomatin-induced defects. These data suggest that stomatin interfaces with lipid metabolism. Stomatin has multiple contacts with the plasma membrane and we identify which sites are required for its role in cell division, as well as associated lipid shifts. We also show that stomatin's mobility on the plasma membrane changes during division, further supporting the requirement for a highly regulated physical interaction between membrane lipids and this newly identified cell division protein.

Insights Into Mechanisms of Oriented Division From Studies in 3D Cellular Models.

In multicellular organisms, epithelial cells are key elements of tissue organization. In developing tissues, cellular proliferation and differentiation are under the tight regulation of morphogenetic programs, that ensure the correct organ formation and functioning. In these processes, mitotic rates and division orientation are crucial in regulating the velocity and the timing of the forming tissue. Division orientation, specified by mitotic spindle placement with respect to epithelial apico-basal polarity, controls not only the partitioning of cellular components but also the positioning of the daughter cells within the tissue, and hence the contacts that daughter cells retain with the surrounding microenvironment. Daughter cells positioning is important to determine signal sensing and fate, and therefore the final function of the developing organ. In this review, we will discuss recent discoveries regarding the mechanistics of planar divisions in mammalian epithelial cells, summarizing technologies and model systems used to study oriented cell divisions in vitro such as three-dimensional cysts of immortalized cells and intestinal organoids. We also highlight how misorientation is corrected in vivo and in vitro, and how it might contribute to the onset of pathological conditions.

Cellular imaging using emission-tuneable conjugated polymer nanoparticles.

New materials that exhibit tuneable optical properties, notable emission across the visible spectrum, are of immense interest to biologists as they present a broad palette of colours from a single imaging agent that can be utilised in biological detection. Such a flexible system, when combined with the advantages of using conjugated polymer nanoparticles in cell imaging results in a widely useful medical diagnostic system. Here, we describe tuneable emission observed through oxidation of a conjugated polymer followed by the formation of nanoparticles and their subsequent use in cell imaging.

Capping protein regulates actin dynamics during cytokinetic midbody maturation.

During cytokinesis, a cleavage furrow generated by actomyosin ring contraction is restructured into the midbody, a platform for the assembly of the abscission machinery that controls the final separation of daughter cells. The polymerization state of F-actin is important during assembly, ingression, disassembly, and closure of the contractile ring and for the cytoskeletal remodeling that accompanies midbody formation and progression to abscission. Actin filaments must be cleared from the abscission sites before the final cut can take place. Although many conserved proteins interact with and influence the polymerization state of actin filaments, it is poorly understood how they regulate cytokinesis in higher eukaryotes. We report here that the actin capping protein (CP), a barbed end actin binding protein, participates in the control of actin polymerization during later stages of cytokinesis in human cells. Cells depleted of CP furrow and form early midbodies, but they fail cytokinesis. Appropriate recruitment of the ESCRT-III abscission machinery to the midbody is impaired, preventing the cell from progressing to the abscission stage. To generate actin filaments of optimal length, different actin nucleators, such as formins, balance CP's activity. Loss of actin capping activity leads to excessive accumulation of formin-based linear actin filaments. Depletion of the formin FHOD1 results in partial rescue of CP-induced cytokinesis failure, suggesting that it can antagonize CP activity during midbody maturation. Our work suggests that the actin cytoskeleton is remodeled in a stepwise manner during cytokinesis, with different regulators at different stages required for successful progression to abscission.

Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A.

Ribonuclease A (RNase A) is widely used in molecular biology research both for analytical assays and for nucleic acid preparation. The catalytic mechanism of RNase A is well understood and absolutely precludes activity on DNA; however anecdotal reports of DNA degradation by RNase A are not uncommon. Here we describe a mechanism by which RNase A treatment can lead to apparent DNA degradation. This results from the surprising finding that RNase A remains functional in a phenol:chloroform mixture, to our knowledge the only enzyme that survives this highly denaturing solvent environment. Although RNase A does not cleave the DNA backbone it is capable of binding to DNA, forming stable RNase A-DNA complexes that partition to the interphase or organic phase during phenol:chloroform purification. The unexpected survival of the RNase A DNA-binding activity in phenol means that these complexes are not dissolved and a substantial amount of RNase A-bound DNA is permanently removed from the aqueous phase and lost on phase separation. This effect will impact DNA recovery from multiple procedures and is likely to represent a source of sequence bias in genome-wide studies. Our results also indicate that the results of analytical studies performed using RNase A must be considered with care.