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Nuclear pore complexes (NPCs) are ∼100 MDa transport channels assembled from multiple copies of ∼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture. In addition to forming the permeability barrier, FG repeats in nucleoporins contribute structurally to nuclear pore biogenesis and function. Keywords: Nuclear pore complex; Nuclear pore biogenesis; Nuclear pore structure; Intrinsically disordered domains; FG repeats; Nuclear envelope; Protein interactions, National Institutes of Health (U.S.) (Grant R01GM077537)

Direct Numerical Simulations (DNSs) were conducted in a turbulent rough wall pipe with three-dimensional sinusoidal roughness using a conforming grid. The wealth of information provided by these simulations is used to gain a better understanding of rough wall turbulent flows from both a practical and fundamental point of view. A comprehensive range of simulations were conducted where the wavelength and roughness semi-amplitude of the roughness elements are systematically investigated. Two roughness parameters were investigated, namely the average roughness height k_a+ and the effective slope ES. An empirical roughness function model was then developed based on these roughness parameters and is found to reasonably estimate the drag of a variety of rough surfaces even in the transitionally rough regime. The shape, inclination angle of the turbulent structures and the dominant energetic length scales in the flow was found to be affected by ES. In addition, ES can have a dominant influence on the turbulent flow in the fully rough regime and dictate the degree to which outer-layer similarity is observed. For all of the rough cases tested, Townsend's outer-layer similarity is observed when considering the mean and higher-order statistics. There is also good collapse in the contours of the streamwise premultiplied energy spectra of the rough cases with the smooth wall in the outer region of the flow. Despite the large blockage ratio of the largest roughness, good collapse is observed in the outer-layer due to the symmetry of an internal flow where the boundary layer thickness is fixed and the shape of the total stress profile is well-defined. Finally, a rough wall turbulent flow is simulated in the minimal-span channel. This proof of concept shows that accurate and direct determination of drag is obtainable at a fraction of the computational resources required for a full-span channel.

This paper reports design, fabrication, and characterization of an air-actuated microvalve and a micropump made of thermoplastic materials. The bonding process was carried out by thermal fusion process with no particular surface treatment. The developed microvalve was used as a reversible switch for controlling both liquid flow and electrical field. Bonding strength of the fabricated microvalves could withstand liquid and air pressures of up to 600 kPa with no burst failure. The micropump made of three connected microvalves, actuated by compressed air, could generate a liquid flow rate of up to 85 µl/min. The proposed microvalve and micropump can be used as pre-fabricated off-the-shelf microfluidic functional elements for easy and rapid integration with thermoplastic microfluidic circuitries in a plug-and-play arrangement.

In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function., National Science Foundation (U.S.) (Predoctoral Fellowship), National Institutes of Health (U.S.) (grant GM056800), Howard Hughes Medical Institute (Investigator)

How spatial information is translated into a chemical signal is a fundamental problem in all organisms. The spindle position checkpoint is a prime example of this problem. This checkpoint senses spindle position and, in budding yeast, inhibits the mitotic exit network (MEN), a signaling pathway that promotes exit from mitosis. We find that spindle position is sensed by a system composed of MEN-inhibitory and -activating zones and a sensor that moves between them. The MEN inhibitory zone is located in the mother cell, the MEN-activating zone in the bud, and the spindle pole body (SPB), where the components of the MEN reside, functions as the sensor. Only when an SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud where the MEN activator Lte1 resides can exit from mitosis occur. In this manner, spatial information is sensed and translated into a chemical signal., National Science Foundation (U.S.) (NSF Predoctoral Fellowship), Howard Hughes Medical Institute (Investigator), National Institutes of Health (U.S.) (GM056800)

For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother–bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell., Howard Hughes Medical Institute, National Science Foundation (U.S.) (Predoctoral Fellowship), National Institutes of Health (U.S.) (Grant GM056800)

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010., Cataloged from PDF version of thesis., Includes bibliographical references., Most cells are polarized in that they are aware of spatial cues and can respond to these cues accordingly. One major aspect of cell function that is often responsive to these polarization cues is cell division. Cell division, the process of making two cells from one progenitor, requires equal distribution of the genetic material to the two progeny cells. When polarized cells divide, an additional constraint on the segregation of the genetic material is imposed, namely, cells must divide the genetic material along axes defined by polarization cues. In eukaryotes, this problem is generally solved by the positioning of the mitotic spindle according to these spatial cues. Defects in spindle positioning can lead to the generation of cells with incorrect organelle, genetic and molecular contents, fate and/or, spatial orientation. Cells have evolved feedback mechanisms that monitor defects in spindle positioning and delay the cell cycle in response to such defects. These mechanisms are best elucidated in the budding yeast, Saccharomyces cerevisiae. The protein kinase Kin4 inhibits the Mitotic Exit Network when the spindle is mis-positioned. How Kin4 is itself regulated and whether or how Kin4 responds to spindle mis-position is not known. The work presented in this thesis elucidates the regulation of Kin4. We identify a novel spindle position checkpoint component, PP2A-Rts 1, and show that it promotes checkpoint function by enabling proper Kin4 localization. We also identify domains and sequence determinants within Kin4 that control localization and function. We present a model of how the spindle position checkpoint senses spindle position and test this model for Kin4 function. We find that the generation of positive and negative mitotic exit regulatory zones allows the cell to sense and translate the spatial information of spindle position into a chemical cell cycle signal., by Leon Y. Chan., Ph.D.

This document is an prior version of the manuscript 'Refactoring Bacteriophage T7' that is now published in Nature/EMBO Molecular Systems Biology (DOI: 10.1038/msb4100025). This DSpace manuscript is more concise but provides less context than the MSB manuscript., Natural biological systems are selected by evolution to continue to exist. Evolution might give rise to complicated systems that are difficult to discover, measure, model, and direct. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered alternative that is easier to study, understand, and extend. We replaced the left 11,515 base pairs of the wild-type genome with 12,179 base pairs of redesigned DNA. The resulting chimeric genome encodes a viable bacteriophage that maintains key features of the original while being simpler to model and easier to manipulate.