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An improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, is introduced, called hmKeima8.5. Its increased intracellular brightness and long Stokes shift (~180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Here we demonstrate its utility in intracellular fluorescence correlation spectroscopy applications.

The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a "wait anaphase" signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31comet, a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31cometduring normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31comettraffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31cometarrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31cometis required for timely mitotic exit. We propose that p31cometis an essential component of the machinery that silences the checkpoint during each cell cycle.

It has been a significant challenge to quantitatively study the dynamic intracellular processes in live cells. These studies are essential for a thorough understanding of the underlying mechanisms regulating the signaling pathways and the transitions between cell cycle stages. Our studies of Cdc20, an important mitotic checkpoint protein, throughout the cell cycle demonstrate that fluorescence correlation spectroscopy is a powerful tool for in vivo quantitative studies of dynamic intracellular processes. In this study, Cdc20 is found to be present primarily in a large complex (>1Mda) during interphase with a diffusion constant of 1.8±0.1μm2/s and a concentration of 76±24nM, consistent with its association with the APC/C. During mitosis, however, a proportion of Cdc20 dissociates from APC/C at a rate of 12pM/s into a soluble pool with a diffusion constant of 19.5±5.0μm2/s, whose size is most consistent with free Cdc20. This free pool accumulates to 50% of total Cdc20 (∼40nM) during chronic activation of the mitotic checkpoint but disappears during mitotic exit at a rate of 31pM/s. The observed changes in the biochemical assembly states of Cdc20 closely correlate to the known temporal pattern of the activity of APC/CCdc20 in mitosis. Photon counting histograms reveal that both complexes contain only a single molecule of Cdc20. The underlying mechanisms of the activities of APC/CCdc20 throughout the cell cycle are discussed in light of our experimental observations.

This study investigates the feasibility of using the enhanced cyan mutant of green fluorescent protein (ECFP) as a probe for two-photon fluorescence correlation spectroscopy (FCS). Molecular dynamics and other properties of ECFP and an ECFP-tubulin fusion protein were investigated in living Potorous tridactylis(PTK2) cells. ECFP has high molecular brightness in the nucleus (=3.3 kcpsm)and in the cytoplasm (3.2 kcpsm) under our experimental conditions. The diffusion constants of ECFP were determined to be 20±7 m2/sin the nucleus and 21±8 m2/sin the cytoplasm. ECFP has stable molecular characteristics with negligible photobleaching and photodynamic effects in our measurements. At the highest concentration of monomer ECFP (425 nM) the amount of dimer ECFP was estimated to be negligible (∼1.8 nM), consistent with our data analysis using a single species model. ECFP-tubulin has a diffusion constant of 6 m2/s in the living cells. In addition, we demonstrate that analysis of the molecular brightness can provide a new avenue for studying the polymerization state of tubulin. We suggest that the tubulin in the vicinity of the nucleus exists primarily as a heterodimer subunit while those in the area away from the nucleus (d>5 m)are mostly oligomers. We conclude that ECFP is a useful genetic fluorescent probe for FCS studies of various cellular processes when in fusion to other biomolecules of interest. © 2004 Society of Photo-Optical Instrumentation Engineers.

Neuronal cytoskeletal elements such as neurofilaments, F-actin, and microtubules are actively translocated by an as yet unidentified mechanism. This report describes a novel interaction between neurofilaments and microtubule motor proteins that mediates the translocation of neurofilaments along microtubules in vitro. Native neurofilaments purified from spinal cord are transported along microtubules at rates of 100-1000 nm/s to both plus and minus ends. This motion requires ATP and is partially inhibited by vanadate, consistent with the activity of neurofilament-bound molecular motors. Motility is in part mediated by the dynein/dynactin motor complex and several kinesin-like proteins. This reconstituted motile system suggests how slow net movement of cytoskeletal polymers may be achieved by alternating activities of fast microtubule motors.

Introduction:The ability to deliver genes to specific cell types in vivowould have a profound therapeutic impact for a diverse set of diseases. For example, targeting T cells for in vivodelivery of a chimeric antigen receptor (CAR) to treat B cell malignancies would improve access to CAR T therapies by overcoming the limitations of ex vivomanufacturing such as high costs, wait times and manufacturing failures. We have developed a novel paramyxovirus-based integrating vector (fusosomes) that specifically targets cell surface receptors for targeted gene delivery. Fusosomes, engineered to target CD8α, a cell surface protein expressed on CD8+ T cells, can bind and specifically deliver a genetic payload through membrane fusion. To evaluate the specificity of fusosome-mediated delivery to cells expressing CD8α in vitro, single cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (scTCR-seq) were performed on human PBMCs treated with CD8α-targeted fusosomes with a GFP payload. scRNA-seq is a tool that can be used to detect the transgene delivered by our fusosomes in specific cell populations by measuring mRNA expression of the receptor targeted by the fusosome (e.g., CD8α) and the genetic payload delivered by the fusosome in the same cell. Transcriptome information to understand potential pathway changes induced by delivery of the transgene is also captured.

Introduction: Ex vivomanufactured chimeric antigen receptor (CAR) T cell therapies are highly effective for treating B cell malignancies. However, the complexity, cost and time required to manufacture CAR T cells limits access. To overcome conventional ex vivoCAR T limitations, a novel gene therapy platform has been developed that can deliver CAR transgenes directly to T cells through systemic administration of a fusosome, an engineered, target-directed novel paramyxovirus-based integrating vector that binds specific cell surface receptors for gene delivery through membrane fusion. Here, we demonstrate that systemic administration of a CD8a-targeted, integrating vector envelope (i.e., fusogen) encoding an anti-CD20 CAR into Southern pig-tail macaques (M. nemestrina), which is a species permissive to the integrating vector-mediated transduction, results in T cell transduction and B cell depletion with no treatment-related toxicities.

Introduction:Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivomanipulation of T cells is likely to have a negative impact on quality. In vivogene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells.

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Biological macromolecules have unique rheological properties that distinguish them from common synthetic polymers. Among these, fibrin has been studied extensively to understand the basic mechanisms of viscoelasticity as well as molecular mechanisms of coagulation disorders. One aspect of fibrin gel rheology that is not observed in most polymeric systems is strain hardening: an increase in shear modulus at strain amplitudes above 10%. Fibrin clots and plasma clots devoid of platelets exhibit shear moduli at strains of approximately 50% that are as much as 20 times the moduli at small strains. The strain hardening of fibrin gels was eliminated by the addition of platelets, which caused a large increase in shear storage modulus in the low strain linear viscoelastic limit. The reduction in strain hardening may result from fibrin strand retraction which occurs when platelets become activated. This interpretation is in agreement with recent theoretical treatments of semi-flexible polymer network viscoelasticity.

Comment on: Delaval B, et al. Cell Cycle 2011; 10:3964-72.