mRNP granules. Assembly, function, and connections with disease.

Messenger ribonucleoprotein (mRNP) granules are dynamic, self-assembling structures that harbor non-translating mRNAs bound by various proteins that regulate mRNA translation, localization, and turnover. Their importance in gene expression regulation is far reaching, ranging from precise spatial-temporal control of mRNAs that drive developmental programs in oocytes and embryos, to similarly exquisite control of mRNAs in neurons that underpin synaptic plasticity, and thus, memory formation. Analysis of mRNP granules in their various contexts has revealed common themes of assembly, disassembly, and modes of mRNA regulation, yet new studies continue to reveal unexpected and important findings, such as links between aberrant mRNP granule assembly and neurodegenerative disease. Continued study of these enigmatic structures thus promises fascinating new insights into cellular function, and may also suggest novel therapeutic strategies in various disease states.

HURP localization in metaphase is the result of a multi-step process requiring its phosphorylation at Ser627 residue.

Faithful chromosome segregation during cell division requires accurate mitotic spindle formation. As mitosis occurs rapidly within the cell cycle, the proteins involved in mitotic spindle assembly undergo rapid changes, including their interactions with other proteins. The proper localization of the HURP protein on the kinetochore fibers, in close proximity to chromosomes, is crucial for ensuring accurate congression and segregation of chromosomes. In this study, we employ photoactivation and FRAP experiments to investigate the impact of alterations in microtubule flux and phosphorylation of HURP at the Ser627 residue on its dynamics. Furthermore, through immunoprecipitations assays, we demonstrate the interactions of HURP with various proteins, such as TPX2, Aurora A, Eg5, Dynein, Kif5B, and Importin ß, in mammalian cells during mitosis. We also find that phosphorylation of HURP at Ser627 regulates its interaction with these partners during mitosis. Our findings suggest that HURP participates in at least two distinct complexes during metaphase to ensure its proper localization in close proximity to chromosomes, thereby promoting the bundling and stabilization of kinetochore fibers.

The stability of NPM1 oligomers regulated by acidic disordered regions controls the quality of liquid droplets.

The nucleolus is a membrane-less nuclear body that typically forms through the process of liquid-liquid phase separation (LLPS) involving its components. NPM1 drives LLPS within the nucleolus and its oligomer formation and inter-oligomer interactions play a cooperative role in inducing LLPS. However, the molecular mechanism underlaying the regulation of liquid droplet quality formed by NPM1 remains poorly understood. In this study, we demonstrate that the N-terminal and central acidic residues within the intrinsically disordered regions (IDR) of NPM1 contribute to attenuating oligomer stability, although differences in the oligomer stability were observed only under stringent conditions. Furthermore, the impact of the IDRs is augmented by an increase in net negative charges resulting from phosphorylation within the IDRs. Significantly, we observed an increase in fluidity of liquid droplets formed by NPM1 with decreased oligomer stability. These results indicate that the difference in oligomer stability only observed biochemically under stringent conditions has a significant impact on liquid droplet quality formed by NPM1. Our findings provide new mechanistic insights into the regulation of nucleolar dynamics during the cell cycle.

3,5-bis(styryl)pyrazole inhibits mitosis and induces cell death independent of BubR1 and p53 levels by depolymerizing microtubules.

Here, we show that 3,5-bis[(1E)-2-(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l depolymerizes microtubules and reduces the number of growing tips of microtubules. The fluorescence recovery after photobleaching experiment in live MCF-7 cells showed that pyrazole 2l suppresses spindle microtubule dynamics. Further, the compound inhibits chromosome movements, activates the spindle assembly checkpoint and blocks mitosis in MCF-7 cells. Pyrazole 2l treatment induced cell death in a variety of pathways. Pyrazole 2l induces cell death independent of BubR1 and p53 levels of MCF-7 cells upon microtubule depolymerization. Further, pyrazole 2l increases the interaction between NF-κB and microtubules and enhances the nuclear localization of NF-κB at its half-maximal proliferation inhibitory concentration while a high concentration of the compound reduced the nuclear localization of NF-κB. Interestingly, the compound exerted significantly stronger antiproliferative effects in cancerous cells than in non-cancerous cells. The results indicated that pyrazole 2l inhibits mitosis by targeting microtubules, induces several types of cell death stimuli and suggests its potential as a lead in developing anticancer agent.

Localization of drug biodistribution in a 3D-bioengineered subcutaneous neovascularized microenvironment.

Local immunomodulation has shown the potential to control the immune response in a site-specific manner for wound healing, cancer, allergy, and cell transplantation, thus abrogating adverse effects associated with systemic administration of immunotherapeutics. Localized immunomodulation requires confining the biodistribution of immunotherapeutics on-site for maximal immune control and minimal systemic drug exposure. To this end, we developed a 3D-printed subcutaneous implant termed 'NICHE', consisting of a bioengineered vascularized microenvironment enabled by sustained drug delivery on-site. The NICHE was designed as a platform technology for investigating local immunomodulation in the context of cell therapeutics and cancer vaccines. Here we studied the ability of the NICHE to localize the PK and biodistribution of different model immunomodulatory agents in vivo. For this, we first performed a mechanistic evaluation of the microenvironment generated within and surrounding the NICHE, with emphasis on the parameters related to molecular transport. Second, we longitudinally studied the biodistribution of ovalbumin, cytotoxic T lymphocyte-associated antigen-4-Ig (CTLA4Ig), and IgG delivered locally via NICHE over 30 days. Third, we used our findings to develop a physiologically-based pharmacokinetic (PBPK) model. Despite dense and mature vascularization within and surrounding the NICHE, we showed sustained orders of magnitude higher molecular drug concentrations within its microenvironment as compared to systemic circulation and major organs. Further, the PBPK model was able to recapitulate the biodistribution of the 3 molecules with high accuracy (r â€‹> â€‹0.98). Overall, the NICHE and the PBPK model represent an adaptable platform for the investigation of local immunomodulation strategies for a wide range of biomedical applications.

Off the wall: The rhyme and reason of Neurospora crassa hyphal morphogenesis.

The fungal cell wall building processes are the ultimate determinants of hyphal shape. In Neurospora crassa the main cell wall components, ß-1,3-glucan and chitin, are synthesized by enzymes conveyed by specialized vesicles to the hyphal tip. These vesicles follow different secretory routes, which are delicately coordinated by cargo-specific Rab GTPases until their accumulation at the Spitzenkörper. From there, the exocyst mediates the docking of secretory vesicles to the plasma membrane, where they ultimately get fused. Although significant progress has been done on the cellular mechanisms that carry cell wall synthesizing enzymes from the endoplasmic reticulum to hyphal tips, a lot of information is still missing. Here, the current knowledge on N. crassa cell wall composition and biosynthesis is presented with an emphasis on the underlying molecular and cellular secretory processes.

Theoretical model of mitotic spindle microtubule growth for FRAP curve interpretation.

BACKGROUND: Spindle FRAP curves depend on the kinetic parameters of microtubule polymerization and depolymerization. The empirical FRAP curve proposed earlier permits determination of only one such dynamic parameter, commonly called the "tubulin turnover". The aim of our study was to build a FRAP curve based on an already known kinetic model of microtubule growth. RESULTS: A numerical expression that describes the distribution of polymerizing and depolymerizing microtubule ends as a function of four kinetic parameters is presented. In addition, a theoretical FRAP curve for the metaphase spindle is constructed using previously published dynamic parameters. CONCLUSION: The numerical expression we elaborated can replace the empirical FRAP curve described earlier for a spindle comprising fluorescently marked microtubules. The curve we generated fits well the experimental data.

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis.

To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.

Generation of an alpaca-derived nanobody recognizing γ-H2AX.

Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers.

Arp2/3 complex function in the epidermis.

An exciting frontier in biology is understanding the functions of basic cell biological machinery in complex tissues. This approach is expected to uncover novel modes of regulation as well as reveal how core machinery is repurposed by different tissues to accomplish different physiological outputs. F-actin plays roles in cell shape, adhesion, migration and signaling - diverse functions that require a specific organization established by a myriad of regulators. Here, we discuss the role of the actin nucleating Arp2/3 complex and the unexpected roles that it plays in a stratified epithelial tissue, the epidermis. While many expected phenotypes such as defects in architecture and cell adhesion were lacking, loss of the Arp2/3 complex activity resulted in epidermal barrier and differentiation defects. This teaches us that, while informative, cell culture approaches are limiting and that studies of the Arp2/3 complex in diverse tissues are expected to yield many more surprises.