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All materials enter or exit the cell nucleus through nuclear pore complexes (NPCs), efficient transport devices that combine high selectivity and throughput. NPC-associated proteins containing phenylalanine–glycine repeats (FG nups) have large, flexible, unstructured proteinaceous regions, and line the NPC. A central feature of NPC-mediated transport is the binding of cargo-carrying soluble transport factors to the unstructured regions of FG nups. Here, we model the dynamics of nucleocytoplasmic transport as diffusion in an effective potential resulting from the interaction of the transport factors with the flexible FG nups, using a minimal number of assumptions consistent with the most well-established structural and functional properties of NPC transport. We discuss how specific binding of transport factors to the FG nups facilitates transport, and how this binding and competition between transport factors and other macromolecules for binding sites and space inside the NPC accounts for the high selectivity of transport. We also account for why transport is relatively insensitive to changes in the number and distribution of FG nups in the NPC, providing an explanation for recent experiments where up to half the total mass of the FG nups has been deleted without abolishing transport. Our results suggest strategies for the creation of artificial nanomolecular sorting devices.
Author Summary
The DNA at the heart of our cells is contained in the nucleus. This nucleus is surrounded by a barrier in which are buried gatekeepers, termed nuclear pore complexes (NPCs), which allow the quick and efficient passage of certain materials while excluding all others. It has long been known that materials must bind to the NPC to be transported across it, but how this binding translates into selective passage through the NPC has remained a mystery. Here we describe a theory to explain how the NPC works. Our theory accounts for the observed characteristics of NPC–mediated transport, and even suggests strategies for the creation of artificial nanomolecular sorting devices.
Citation: Zilman A, Di Talia S, Chait BT, Rout MP, Magnasco MO (2007) Efficiency, Selectivity, and Robustness of Nucleocytoplasmic Transport. PLoS Comput Biol 3(7): e125. https://doi.org/10.1371/journal.pcbi.0030125
Editor: Susan Wente, Vanderbilt University, United States of America
Received: August 16, 2006; Accepted: May 17, 2007; Published: July 13, 2007
Copyright: © 2007 Zilman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors received no specific funding for this study.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: FG repeats, phenylalanine–glycine repeats; FG nups, a class of NPC-associated proteins containing FG repeats; NPC, nuclear pore complex
The contents of the eukaryotic nucleus are separated from the cytoplasm by the nuclear envelope. Nuclear pore complexes (NPCs) are large protein assemblies embedded in the nuclear envelope and are the sole means by which materials exchange across it. Water, ions, small macromolecules (<40 kDa) [1], and small neutral particles (diameter <5 nm) can diffuse unaided across the NPC [2], while larger macromolecules (and even many small macromolecules) will generally only be transported efficiently if they display a particular transport signal sequence, such as a nuclear localization signal (NLS) or nuclear export signal (NES). Macromolecular cargoes carrying these signal sequences bind cognate soluble transport factors that facilitate the passage of the resulting transport factor–cargo complexes through the NPC. The-best studied transport factors belong to a family of structurally related proteins, collectively termed β-karyopherins, although other transport factors can also mediate nuclear transport, particularly the export of mRNAs (reviewed in [1,3–6]). NPCs can pass cargoes up to 30 nm diameter (such as mRNA particles), at rates as high as several hundred macromolecules per second—each transport factor–cargo complex dwelling in the NPC for a time on the order of 10 ms [7,8].
Here we focus on karyopherin-mediated import, although our conclusions pertain to other types of nucleocytoplasmic transport as well, including mRNA export. During import, karyopherins bind cargoes in the cytoplasm via their nuclear localization signals. The karyopherin–cargo complexes then translocate through NPCs to the nucleoplasm, where the cargo is released from the karyopherin by RanGTP, which is maintained in its GTP-bound form by a nuclear factor, RanGEF. The high affinity of RanGTP binding for karyopherins allows it to displace cargoes from the karyopherins in the nucleus. Subsequently, karyopherins with bound RanGTP travel back through the NPC to the cytoplasm, where conversion of RanGTP to RanGDP is stimulated by the cytoplasmic factor RanGAP. The energy released by GTP hydrolysis is used to dissociate RanGDP from the karyopherins, which are then ready for the next cycle of transport. Importantly, this GTP hydrolysis is the only step in the process of nuclear import that requires an input of metabolic energy. Overall, the energy obtained from RanGTP hydrolysis is used to create a concentration gradient of karyopherin–cargo complexes between the cytoplasm and the nucleus, so that the process of actual translocation across the NPC occurs purely by diffusion [1,3–6,9–15].
Conceptually, nuclear import can be divided into three stages: first, the loading of cargo onto karyopherins in the cytoplasm, second, the translocation of karyopherin–cargo complexes through the NPC, and, third, the release of cargo inside the nucleus (Figure 1). The first and last stages have been the subject of numerous studies, and are relatively well understood, being soluble-phase reactions amenable to biochemical characterization (reviewed in [1,3–6,15]). The intermediate stage of transport is much less understood. Nevertheless, it is clear that the ability of karyopherins (and other transport factors) to bind a particular class of NPC-associated proteins containing phenylalanine–glycine (FG) repeats, known collectively as FG nups, is a key feature of the transport process, and allows them to selectively and efficiently pass with their cargoes through the NPC. In particular, experiments in which the FG nup–binding sites on the karyopherins were mutated show that disrupting the binding of karyopherins to FG nups impairs transport [6,12,13,15,16]. Current estimates of the binding affinity of karyopherins to most FG nups are in the range 1–1,000 nM (or 10–30 kBT per binding site), depending on the FG nup and karyopherin type [18–20]. Each FG nup usually carries a small region that anchors it to the body of the NPC, and a larger region characterized by multiple FG repeats. These FG repeat regions are natively disordered flexible chains or filaments that contain binding sites for transport factors (including karyopherins) and also appear to set up a barrier at the entrance of the NPC for macromolecules that cannot bind them [1,3,4,9,13,14,21–23]. The detailed physicochemical nature of this barrier is still under active study, although FG nups have been shown in vitro to form flexible polymer brushes when grafted to a surface [22] or gels in bulk solution [24]. Importantly, it has been repeatedly demonstrated that individual FG repeat regions can have a long reach, on the order of many tens of nm, within the NPC [3,22,23,25]. What is still needed is a quantitative theoretical explanation that can account for the observed characteristics of facilitated nuclear–cytoplasmic transport.
(A) Schematic of the nuclear import process. The karyopherins bind the cargo in the cytoplasm and transport it to the nucleus, where the cargo is released by RanGTP.
(B) Schematic of the NPC. The nucleus and the cytoplasm are connected by a channel, which is filled with flexible, mobile filamentous proteins termed FG nups. The karyopherins carrying a cargo enter from the cytoplasm and hop between the binding sites on the FG nups until they either reach the nuclear side of the NPC or return to the cytoplasm.
Here, we develop a diffusion-based theory to explain the mechanism of the intermediate stage of nucleocytoplasmic transport—i.e., translocation through the NPC. A useful theory of NPC-mediated transport should provide insight into several major unresolved questions, including: (i) How does the NPC achieve high transport efficiency of cargoes of variable sizes and in both directions, through only diffusion of the transport factor–cargo complexes? (ii) How does binding of transport factors to FG nups facilitate transport efficiency while maintaining a high throughput (up to hundreds of molecules per second per NPC) [7,8,12,13,26]? (iii) NPCs largely exclude nonspecific macromolecules in favor of transport factor–bound cargoes (reviewed in e.g., [4]). How is this high degree of selectivity achieved? (iv) Neither deletion of up to half the mass of the FG nups' filamentous unfolded regions, nor deletion of asymmetrically disposed FG nups' filamentous regions that potentially set up an affinity gradient, abolish transport [27]. Directionality of transport across the NPC can even be reversed by reversing the concentration gradient of RanGTP [28]. How can we account for such a high degree of robustness?
Several theoretical models have been proposed for the mechanism of transport through the NPC. These include the Brownian Affinity Gate model [4,14], Selective Phase models [12,13,29,30], the Oily Spaghetti model [1], Affinity Gradient models [10,11,15,20,31], the Dimensionality Reduction model [32], and most recently a Two-Gate model [48]. All these models can be thought of as viewing the NPC as a "Virtual Gate" [4,14], where the FG nups set up a barrier for entrance into the NPC and transport through the NPC involves facilitated diffusion controlled by association and disassociation of transport receptors with FG nups. They differ only in specific assumptions, such as the conformation and spatial deployment of the FG nups, their physicochemical state, or the distribution of affinities of binding sites (reviewed in [6]).
The aim of the present paper is to establish a general quantitative framework for NPC transport that is consistent with well-established structural and functional properties of the NPC and its components. We explain how the binding of karyopherins to the FG nups' flexible filaments inside the NPC can give rise to efficient transport. We demonstrate that competition for the limited space and binding sites within the NPC leads to a novel, highly selective filtering process. Finally, we explain how the flexibility of the FG nups could account for the high robustness of NPC-mediated transport with respect to structural changes [27]. We conclude by discussing verifiable experimental predictions of the model.
Setting Up a Physical Model of NPC Transport
The NPC contains a central channel (approximately 35 nm in diameter) that connects the nucleoplasm with the cytoplasm. The internal volume of this channel, as well as large fractions of the nuclear and cytoplasmic surfaces of the NPC, is occupied by the flexible FG-repeat regions of the FG nups (i.e., that portion in each FG nup containing multiple FG repeats). Since these FG-repeat regions also protrude into the nucleus and the cytoplasm, the effective length of the NPC is estimated to be 70 nm [1,3,4]. The details of the distribution of the FG-repeat regions inside the central channel and the external surfaces of the NPC, as well as the exact number of binding sites on the karyopherins and the number of the FG-repeats on the FG-repeat regions that are accessible for binding, have not yet been well-established (although the number of the FG repeats is in the range of 5–50 per FG nup [6,27]). We made no specific assumptions about the distribution of FG nups, interactions between them, and their density, degree of flexibility, or conformation within the NPC. As we will discuss, the general features of transport through the NPC appear relatively insensitive to these details.
We represent transport through the NPC as a combination of two independent processes contributing to the movement of the karyopherin–cargo complexes through the central channel of the NPC: (i) the binding and unbinding of the karyopherins to the FG-repeat regions, and (ii) the spatial diffusion of the complexes, either in the unbound state or while still bound to a flexible FG-repeat region. The complexes entering the NPC from the cytoplasm thus stochastically hop back and forth inside the channel until they either reach the nuclear side, where the cargo is released by RanGTP, or return to the cytoplasm. Detachment from the FG-repeat regions and exit from the NPC can be either thermally activated, or catalyzed by RanGTP directly at the nuclear exit of the NPC [1,4]. A schematic illustration of transport through the NPC is shown in Figure 1.
Enhanced Transport Efficiency Arises from the Karyopherins' Ability to Bind to FG nups
It is important to distinguish between two different properties of the transport process, namely, (i) the speed with which individual complexes traverse the NPC, and (ii) the probability that complexes, entering from the cytoplasm, arrive at the nuclear side [1,4,9,33–35]. As we discuss below, binding of karyopherins to the FG nups increases the probability of the karyopherins traversing the NPC, i.e., their transport efficiency; in the absence of such binding, the probability of traversing the NPC is low.
For simplicity, we assume that the unbinding and rebinding occur faster than the lateral diffusion of karyopherin–cargo complexes along the channel (although our conclusions were verified by computer simulations for any ratio of binding–unbinding rate to diffusion rate, unpublished data). In this limit, movement through the NPC can be approximated by diffusion in an effective potential as explained below. The strength of the effective potential depends on the relative strength of two effects. The first effect is the entropic repulsion between karyopherin–cargo complexes and FG-repeat regions and between the FG-repeat regions themselves, as the karyopherin–cargo complexes have to compress and displace the FG-repeat region filaments to enter the channel. The second effect is an attraction due to the binding of karyopherin–cargo complexes to the FG-repeat regions, as illustrated in Figure 2.
The NPC channel is represented by a potential well U(x), shown in black. The complexes enter the NPC at x = R at an average rate J. A fraction of the entrance flux, JM, goes through to the nucleus. The rest return to the cytoplasm at an average rate, J0. The exit of the complexes from the channel into the nucleus occurs either due to thermal activation, with the rate JL, or by activated release by RanGTP, with the rate Je. Steady state particle density inside the channel, ρ(x), is shown in blue. It differs from what would be expected from equilibrium statistical mechanics as the complexes do not accumulate at the minimum of the potential but rather their density decreases toward the exit.
We represent the transport of karyopherin–cargo complexes through the NPC as diffusion in a one-dimensional potential, U(x) (expressed in units of kBT), in the interval 0 < x < L (Figure 2). The shape of the potential in the NPC is determined by the distribution of the FG nups along the channel (an issue we address later). The actual length of the NPC corresponds to the interval from x = R to x = L − R, and the regions of length R (on the order of the width of the channel [33,36]) at both ends of the interval correspond to the distance outside the NPC over which the particles diffuse into either the nucleoplasm or the cytoplasm. We did not directly model the diffusion of complexes outside of the NPC. Instead, we assumed that karyopherin–cargo complexes stochastically entered the NPC from the cytoplasm, with an average rate J at x = R, where J is proportional to the concentration of the karyopherin–cargo complexes in the cytoplasm [26,36]. Because of the random nature of movement inside the channel, a certain fraction of the complexes impinging on the channel entrance will not reach the nucleus and eventually return to the cytoplasm. We modeled this event by imposing absorbing boundary conditions at x = 0 and x = L that correspond to a karyopherin–cargo complex returning back to the cytoplasm, or going through to the nucleus, respectively.
Thus, the entrance current, J, splits into J0 and JM, corresponding to the flux of complexes returning to the cytoplasm and going through to the nucleus, respectively. Active release of the karyopherin–cargo complexes from the NPC by the nuclear RanGTP is modeled by imposing an additional exit flux, Je (proportional to the nuclear concentration of RanGTP), at a position x = L − R. Therefore, the transmitted flux, JM, splits into Je and JL, which correspond respectively to the flux of karyopherin–cargo complexes released from the FG-repeat regions by RanGTP and to thermally activated release, as shown in Figure 2.
The efficiency of the transport through the NPC is determined by the fraction of the complexes that reach the nucleus, JM/J. We emphasize that we did not study the equilibrium thermodynamic properties of the channel, but rather the steady state, out-of-equilibrium behavior.
We neglected possible differences in the diffusion coefficient of the complexes inside and outside the NPC to focus on the role of karyopherin binding in the import process. We also assumed that no current enters the NPC from the nucleus as the cargoes are released from the karyopherins in the nucleus by RanGTP. Finally, we neglected variations of the potential in the direction perpendicular to the channel axis. The effects of these factors do not change our conclusions, and will be studied in detail elsewhere.
Under the above assumptions, the model can be solved using standard theory of stochastic processes [34]. Importantly, the model can be solved for a potential of an arbitrary shape, allowing us to model different distributions of binding sites within the NPC.
The transport of the karyopherin–cargo complexes through the NPC was then described by the diffusion equation for the density of complexes inside the channel, ρ(x) where the local flux of the complexes within the NPC, J(x), is given by: The first term in Equation 2 describes the random thermal motion of the complexes, and the second term stands for the variations in the flux due to local variations of the potential U(x); D is the diffusion coefficient of the complexes inside the channel.
The steady state density of the complexes in the channel, obtained by solving Equations 1 and 2, with entrance flux J and satisfying the boundary conditions ρ(0) = ρ(L) = 0, is
The sum of the flux of karyopherin–cargo complexes going through the NPC, and of that returning to the cytoplasm, is equal to the total flux of complexes entering the NPC; hence |J0| + JM = J; similarly, JM − JL = Je. The flux, Je, is proportional to the number of complexes present at the nuclear exit, and to the frequency, Jran, with which RanGTP molecules hit the nuclear exit of the NPC: Je = Jranρ(x = L −R)R. Recalling that the potential outside the channel is zero (U(x) = 0) for 0 < x < R and L − R < x < L, and using the continuity of ρ(x) at x = L − R, one obtains for Ptr, the probability of a given karyopherin–cargo complex reaching the nucleus (i.e., the fraction of complexes reaching the nucleus): where K = JranR2/D exp(−U(L−R)).
Equation 4 is the main result of this section and has several important consequences. The probability of traversing the NPC, Ptr, defines the transport efficiency. This efficiency is seen to increase with the potential depth E, (defined as E = −minxU(x), Figure 2), proportional to the binding strength of the karyopherin–cargo complexes to the FG-repeat regions. In the absence of binding, Ptr is small (∼R/L), so that a complex will, on average, return to the cytoplasm soon after entering the NPC. Notably, an attractive potential inside the NPC increases the time the complex spends inside the NPC and thus increases the probability that it reaches the nuclear side, rather than returns to the cytoplasm.
When RanGTP only releases cargo from its karyopherin, but not from the FG-repeat regions (i.e., Je = 0); the maximal translocation probability, Ptr, is 0.5. However, in the case when RanGTP also releases karyopherin–cargo complexes from FG-repeat regions, the translocation probability, Ptr, can reach unity. Importantly, the latter effect is more pronounced for a large K, that is, for strong binding at the exit. We shall discuss the practical implications of this result later.
The second important consequence of Equation 4 is that Ptr depends only weakly on the shape of the potential, U(x). This can account for why the transport properties of the NPC are relatively insensitive to the details of the distribution of FG-repeat regions inside the NPC, and to the distribution of the binding sites on the FG-repeat regions.
A Mechanism for Selectivity is Provided by the Limited Space and Binding Capacity within the NPC
The previous section does not take into account the interference between karyopherin–cargo complexes inside the channel. Although a large interaction strength, E, increases transport efficiency, this increase is at the expense of an increased transport time T(E), which grows roughly exponentially with E (Text S1), and leads to an accumulation of karyopherin–cargo complexes inside the channel. However, the space and the number of available binding sites inside the channel are limited. As the number of these complexes in the channel increases, they start to interfere with the passage of each other due to molecular crowding. This molecular crowding results from two different sources. One is the repulsion that the entering macromolecules feel from the FG nups that set up the permeability barrier. The second is competition for the limited space inside the channel between the karyopherin–cargo complexes themselves, and which we now demonstrate can determine the selectivity. In effect, these two factors represent the entropic exclusion that we have discussed previously [1,3,4,9,13,14,21–23].
To quantitatively investigate how mutual interference between translocating karyopherin–cargo complexes and molecular crowding affect transport efficiency, we performed dynamic Monte Carlo simulations of the diffusion of complexes inside the NPC, in the potential U(x), using a variant of the Gillespie algorithm [37–39]. The simulations are a discrete version of the continuum formulation of the previous section. The interval [0,L] is represented by N discrete positions, which is a standard way to approximate the continuous diffusion; it is important to appreciate that these sites do not represent the actual binding sites, but correspond to the length of a diffusion step. We allowed only a limited number, nmax, of complexes at each position at any moment of time, which models the competition between complexes for the limited space and the accessible binding sites inside the channel. In line with our analytical model above, karyopherin–cargo complexes were deposited at the position iR if it was unoccupied by a complex, with a probability of JL2/(DN2) per simulation step. When a complex reached position i = 0 (cytoplasm) or i = N (nucleus), it was removed from the channel. In addition, the complexes present at the position i = N − iR could be removed directly, with the probability JranL2/(DN2), which models the effect of the release of the complexes from FG-repeat regions by nuclear RanGTP. Once inside the channel, a complex present at site i could hop to an adjacent unoccupied site, i ± 1, with the following probability: where xi is the site occupancy: xi = 0 if the site is unoccupied, xi = n if n complexes are present at the position i, up to the nmax complexes. The transition rates from a site i to a site i ± 1 were , if xi±1 < nmax, and zero, if xi±1 = nmax [37–39].
The results of our simulations for the experimentally relevant range of interaction strength E and incoming flux J are shown in Figure 3 (see Discussion for an explanation of our choice for the parameter values). Figure 3 shows the results for nmax = 1; the results did not change substantially for higher allowed local occupancy (Text S1 and Figure S5). For low interaction strength, E, the translocation probability curves for all entrance fluxes, J, collapse onto a single line (which is predicted by the analytical solution from the previous section), because in this regime there are few complexes simultaneously present in the channel, and molecular crowding is negligible. For stronger binding, karyopherin–cargo complexes accumulate inside the channel, blocking the inflow of additional complexes, and the channel becomes jammed as reflected in the decrease of the probability to traverse the NPC.
(A) Transport efficiency, as given by the probability to reach the nucleus, is shown as a function of the interaction strength. RanGTP activity in the nucleus is represented by using JranL2/(N2D) = 1.5. The curves correspond to four different values of the entrance rate, J (measured in units of 10−416D/R2); the red line is the low-rate limit of Equation 4. For any entrance rate, the transport efficiency is maximal at a specific value of the interaction strength, which provides a mechanism of selectivity.
(B) Optimal interaction strength of (A) as a function of the incoming rate J (in units of 10−416D/R2), for JranL2/(N2D) = 1.5; parabolic potential shape. See Discussion for the corresponding actual values of the flux through the pore. Black dots are simulation results.
The main conclusion of the simulations, as shown in Figure 3A, is that transport through the NPC is maximal for an optimal value of the interaction strength Ec. Therefore, our theory rigorously confirms the intuitive notion of the existence of an optimal binding that balances increased transport probability with increased time spent within the NPC. In our simulations, the optimal binding strength depends on the entrance flux, and thus on the abundance of complexes of a particular type in the cytoplasm. In particular, the optimal interaction strength is higher for low entrance fluxes, as illustrated in Figure 3B.
Existence of an optimal interaction strength provides a mechanism for the selectivity of NPC-mediated transport. Karyopherin–cargo complexes tuned for a particular strength of interaction with FG-repeat regions have a high translocation probability, while macromolecules that do not interact with FG nups are less likely to cross. We elaborate on this finding in the next section.
Competition between Non-Specifically Binding Macromolecules and Karyopherins Enhances the Selectivity of the NPC
As discussed in the previous section, the binding of karyopherins to FG-repeat regions provides a mechanism of selectivity. However, the maximum depicted in Figure 3A is broad; the translocation probability is significant even for binding strengths considerably lower than the optimal one. For instance, if the optimal interaction strength is E = 15kT (Kd ≈ 300 nM), macromolecules whose interaction strength is 7kT (Kd ≈ 1 mM) [17] have a probability of reaching the nucleus that is more than half the optimal one. On one hand, this broad maximum allows NPC-mediated import to function efficiently across a broad range of transport factor binding strengths. On the other hand, this might also permit passage of macromolecules that bind nonspecifically to FG-repeat regions (e.g., due to electrostatic interactions). However, proper functioning of living cells requires a high selectivity of the NPC—how might this be achieved? So far, Figure 3 only takes into account the competition between complexes of identical binding strength for space inside the channel. However, in a situation where optimally binding karyopherins compete for space and binding sites inside the channel with other, weakly binding macromolecules, the passage of the latter is sharply reduced, which significantly increases the selectivity of the NPC. Qualitatively, because the strongly binding karyopherins and karyopherin–cargo complexes spend more time in the NPC, a weakly binding macromolecule entering the channel will—with high probability—find it occupied by karyopherin–cargo complexes. Therefore, because the residence time of a low affinity macromolecule is relatively short, there is a high probability that it will return to the cytoplasm before the channel clears. On the other hand, if a karyopherin–cargo complex enters a channel that is already occupied by other strongly binding complexes, there is still a high probability that, due to its relatively high residence time, it will reside inside the channel long enough for the complexes that are already inside the NPC to get through. As free karyopherins exchange back and forth across the NPC constantly, there will always be karyopherins (or other transport factors) binding in the NPC and so excluding nonspecific macromolecules, making the NPC a remarkably efficient filter.
These heuristic arguments were verified via computer simulations, using the algorithm of the previous section, adapted to account for two species of particles of different binding strengths. Two species of particles of different binding affinities (representing a karyopherin–cargo complex and another macromolecule that can bind nonspecifically (and weakly) to the FG-repeat regions), are deposited stochastically at the NPC entrance with the same average rate J. As in the previous section, the particles diffuse inside the channel until they either reach the nucleus, or return to the cytoplasm. Due to limited space, each position can be occupied by only a limited number of particles (see Text S2 for the actual code).
As Figure 4 shows, competition for the space inside the channel between the translocating particles dramatically narrows the selectivity curve as compared with Figure 3. This effect is a novel mechanism for the enhancement of transport selectivity beyond what is expected from the equilibrium binding affinity differences alone. In contrast to other mechanisms of specificity enhancement (e.g., kinetic proofreading [40]), no additional metabolic energy is required for this enhanced discrimination. Instead, selectivity is achieved by competition producing a differential NPC response to two ranges of binding affinities. There remains a broad range of higher binding affinities, occupied by transport factors, wherein passage across the NPC is efficient; however, in the low range of affinities, transmission is effectively prevented. We emphasize that this is an essentially nonequilibrium effect, and the selectivity enhancement goes far beyond the difference in the equilibrium binding affinities. Importantly, the enhancement of the selectivity persists even when high local occupancies are allowed (Text S1 and Figure S4).
Shown is the transport efficiency of particles across the NPC as a function of interaction strength with the FG-repeat regions, either in the presence or absence of competing particles. Gray line: transport efficiency of particles as a function of interaction strength in the absence of competition. Red line: transport efficiency of a weakly binding species in an equal mixture of weakly and strongly binding species, as a function of the interaction strength of the weakly binding species; the interaction strength for the strongly binding species is 12kBT. Translocation of the weakly binding species is sharply reduced in the presence of the strongly binding species, until its binding strength approaches that of the strongly binding species. No RanGTP activity was included in these simulations, hence lowering the transport efficiency compared with Figure 3.
The Flexibility of FG-Repeat Regions May Account for the Robustness of NPC-Mediated Transport
In the previous sections, we used a continuous potential to model transport through the NPC. However, in reality the translocating karyopherin–cargo complexes likely hop between discrete binding sites that are located on the separate (or on the same) flexible FG-repeat regions, which fluctuate in space around their anchor points due to thermal motion [3,22,23]. This flexibility allows the complexes to diffuse along the channel while remaining bound to an FG-repeat region. A complex can also unbind from an FG-repeat region and rebind again to the same or a neighboring FG-repeat region, moving while unbound by passive diffusion. In this section, we elucidate how the number of FG nups inside the channel affects transport.
Under these assumptions, the translocation of karyopherin–cargo complexes through the NPC can be described as diffusion in an array of potentials, as illustrated in Figure 5, where each potential well Ui(x) of a width pi represents an FG-repeat region. The shape of each well depends on the number of the binding sites on each FG-repeat region, the binding strength of the karyopherin–cargo complex, and the rigidity and the length of an FG-repeat region, which determine the cost of its entropic stretching in the process of spatial fluctuations. This description allows for the possibility of having several binding sites on an FG nup which affects only the shape of the wells (Text S1 and Figure S1). The blue line in Figure 5 corresponds to the unbound state. Although for the purposes of illustration all the wells are shown to have the same form, the subsequent results are valid for an arbitrary distribution of potential shapes. We shall denote the density of the karyopherin–cargo complexes in the i-th well as ρi(x) and the density of unbound complexes as ρ0(x). The lateral diffusion of the complexes, combined with the binding and unbinding to the FG-repeat regions is then described by the following equations [41]:
Transport through the NPC can be represented as diffusion in an array of potential wells (solid black lines) that represent flexible FG-repeat regions whose fluctuation regions overlap. The red dotted arrows correspond to the complexes unbinding from and rebinding to the FG-repeat regions. The solid blue line represents the unbound state. The solid red line shows the equivalent potential in the case when the unbinding of the complexes from the FG-repeat regions is much faster than the lateral diffusion across an individual well.
The first term in the first equation describes the diffusion in the unbound state, while the second and the third terms describe the unbinding and the binding to the wells. Similarly, the second equation describes the diffusion while still bound to the i-th well (FG-repeat region). The local unbinding and binding rates from the i-th well are ri0(x) and r0i(x), respectively. They are related by the detailed balance condition, r0i(x)/ri0(x) = e−Ui(x)+U0(x), which reflects the energy difference between the bound and unbound states. If the unbinding rates are fast compared with the diffusion time across the wells, the relative densities of bound and unbound complexes are at their local equilibrium Boltzmann ratio [41]: , where ρ(x) = ∑iρi(x) is the total density of the complexes at a position x. Adding up Equations 6, one obtains an equation for the total density of the complexes at position x, ρ(x): where [41]. Thus, the process of translocation through an array of flexible FG nups within the NPC can be described as simple lateral diffusion in the effective potential Ueff, which leads to Equations 1 and 2 in the first section of this manuscript, which are essentially identical to Equation 7, even though they are written in a slightly different form.
As we have proposed in previous sections, the transport properties of the NPC are relatively insensitive to the detailed shape of the effective potential. It follows from Equations 6 and 7 that the shape of the effective potential depends only weakly on the number and the shape of the overlapping potential wells corresponding to the FG-repeat regions.
Even more strikingly, in our model the transport properties of the NPC are not very sensitive to the number of the FG-repeat regions. This is robust with respect to the variations in the number of the FG-repeat regions [27]. This was in striking contrast to the case of the inflexible FG-repeat regions when the binding sites are sparsely distributed without a large degree of overlap (Text S1 and Figure S2). We illustrate this point through a limiting case where the FG-repeat regions barely touch, represented by the potential shown by the blue line in Figure 6A. The flat central part of each potential well corresponds to where the karyopherin–cargo complex is diffusing in the channel while bound to an FG-repeat region, and the sharply rising regions at the borders correspond to unbinding of the complex and its transfer to the next filament. Narrow wells correspond to filaments with limited reach, while wide wells correspond to filaments that can stretch a long distance without significant entropic cost. The potential wells can have different widths, pi, so that their combined width is equal to the total length of the channel, . All the potential wells have the same shape, U0, re-scaled to the width of an individual well, so that the potential at a point x = ∑j<ipj + Δx is: U(x) = U0(Δx/pi).
(A) Effective potential for sparse flexible FG-repeat regions is shown as a blue line. Each well corresponds to an FG-repeat region. The transport properties in this multiwell potential are independent of the number of wells, and hence equivalent to those in the single-well potential, shown as a red line.
(B) Numerical simulations show essentially identical transport efficiencies in the multiwell potential (blue line) and in the single-well potential (red line).
Crucially, the transport properties of the potential shown in Figure 6A do not depend on the number of wells. Both the translocation probability and the residence time are equivalent for the multiwell potential shown in blue, and the single-well potential shown in red, obtained by rescaling an individual blue well to the whole length, L − 2R, of the channel. Indeed, it follows from Equation 4 that the translocation probability for the multiwell potential with n wells, is which is independent of the number and the width of the wells because ∑ipi = L − 2R; as before, K = JranR2/D exp(−U(L − R)). We prove in Text S1 that the residence time is similarly independent of the number of wells. Since both translocation probability and residence time are independent of the number of wells, the transport properties do not depend on the number of wells, even for high entrance rates or binding strengths, when jamming becomes important, as verified by computer simulation (Figure 6B).
This result highlights the robustness of our model of NPC transport; in multiwell potentials of this type, the NPC's transport properties do not depend on the specific number of FG-repeat regions, so long as they are flexible enough for their fluctuation regions to overlap, permitting complexes to freely transfer from one filament to the next, which might explain the puzzling degree of robustness of the NPC transport with respect to the deletion of FG repeat regions [27].
Several conceptual models have been proposed to describe transport through the NPC [1,3–6,9–15,29–32,48]. Most propose that this transport relies on diffusion of the transport factor–cargo complexes in the environment of flexible FG-repeat regions of the FG nups, controlled by transient binding to the FG nups these flexible regions [1,3–6,9–11,14–16].
We have formulated and solved a rigorous mathematical model of transport through the NPC that depends on the physics of diffusion in a channel combined with binding to the flexible filamentous FG-repeat regions (without making detailed assumptions about the conformation and distribution of FG-repeat regions inside the channel). Our model applies to both export and import processes and explains the main features of NPC-mediated transport; namely, its high selectivity for cargoes bound to transport factors, its efficiency and directionality, and its robustness to perturbations.
We propose that the selectivity of the NPC arises from a balance between the probability (efficiency) and the speed of transport of individual karyopherin–cargo complexes. Analogous ideas have been suggested to account for the transport properties of ion channels and porins [14,33–35,42]. In our model, the probability of a karyopherin–cargo complex reaching the nucleus increases with the binding strength of the transport factors to FG-repeat regions, but at the expense of increased residence time inside the NPC; eventually, complexes spend so much time in the NPC that they impede the passage of other complexes through the channel. Therefore, there is an optimal value of the binding strength of karyopherins to FG-repeat regions that maximizes their transport efficiency through the NPC. For karyopherin–cargo complexes with lower entrance fluxes, the optimal binding strength is higher because at low fluxes the accumulating complexes can reside longer in the channel without blocking it (Figure 3). This correlation of optimal binding strength and entrance flux could explain why there are different karyopherin types; the binding strength of each karyopherin type might be related to its cargoes' relative flux and hence the abundance of its cargoes.
By considering known parameters of the nuclear transport machinery, we can test whether our simulations are consistent with the experimentally observed values of the flux through the NPC, and the residence time inside it. We take the effective length of the NPC as L ∼ 70 nm and its effective passive diffusion diameter as R ∼ 7 nm, within the range observed for different NPCs [1]. The diffusion coefficient D of the complexes inside the channel can be estimated to lie in the range 1–10 μ2/s, typical for protein diffusion in the crowded environment of the cytoplasm [43,44]. In vivo, the total cargo flux through an NPC ranges from several molecules per second up to several hundred molecules per second [12,26,45–47]. Accordingly, we performed simulations for values of the incoming flux J in the range 0.3–10 in units of (10−416D/R2), which corresponds to a flux through the NPC in the range of 10–1,000 molecules per second. This results in predicted residence times in the NPC (given by LR/Dexp(E/kT)) of approximately 0.01–1 s, consistent with experimentally determined residence times [7,8]. For incoming flux values in this range, our model predicts optimal interaction strengths in the range of 5–15kBT (Figure 3). Molecular dynamics calculations predict higher binding energies [18], but the effective interaction strength in our model is reduced by entropic effects from the flexible FG-repeat regions.
Our model can also explain the high specificity of facilitated transport through the NPC, wherein each NPC permits the passage of transport factor–cargo complexes but efficiently filters out macromolecules that do not bind specifically to the FG-repeat regions. The difference in binding energy between specifically and nonspecifically binding macromolecules can be as little as a few kBT, which may not seem enough for such efficient discrimination. However, we have uncovered an additional mechanism that we believe significantly enhances the specificity of NPC transport. This mechanism relies on the direct competition between transport factors and nonspecifically binding macromolecules; they compete for space and binding sites in the channel. As a consequence of their stronger binding, transport factors have a longer residence time within the channel as compared with nonspecifically binding macromolecules, which are therefore outcompeted for space and binding sites within the channel. The constant flux of cargo bound or free transport factors between the nucleus and cytoplasm therefore effectively excludes nonspecifically binding macromolecules from the channel. We emphasize that this selectivity enhancement is essentially a nonequilibrium kinetic effect. Hence, although no metabolic energy is expended in this filtering process [40], the resulting selectivity is much higher than might be expected from just the different binding affinities of transport factors and nonspecific macromolecules (Figure 4).
In the case of karyopherin-mediated import, the transport efficiency is enhanced when RanGTP directly releases karyopherins from their binding sites on FG-repeat regions at the NPC exit [1,4,20,31]—an enhancement that increases with the binding strength at the nuclear exit. High affinity binding sites at the nuclear exit of the NPC decrease the probability of return, once a complex has reached the nuclear side. This result may account for the observed high affinity binding sites that are localized at the nuclear side on the NPC in import pathways and at the cytoplasmic side in export pathways [1,4,20,31].
Although the transport properties of the NPC depend strongly on the magnitude of the interaction strengths between transport factors and FG-repeat regions, we predict that transport depends only weakly on spatial variations of the binding strength along the channel. In particular, a gradient of binding affinity across the NPC should not, by itself, increase throughput compared with a uniform distribution of the same sites. This could explain how transport can be reversed across the NPC simply by reversing the gradient of RanGTP [28]. Only a high affinity trap at the exit of the NPC in combination with the action of RanGTP in releasing the karyopherins from this trap can improve the throughput through the NPC.
Although transport relies on the flexibility of the FG-repeat regions, it is relatively insensitive to the number of flexible FG-repeat regions inside the NPC—as long as their fluctuation regions can overlap (Figures 5 and 6). This could account for recent experiments in which up to half the total mass of the flexible FG-repeat regions in NPCs were deleted without abrogating nucleocytoplasmic transport [27]. In particular, it follows from Equation 4 that the probability of traversing the NPC is low if the binding sites are sparse and stationary, unless they are so dense that they occupy almost all the available length of the channel. However, in this case, transport is sensitive to the number of sites. Thus, a theory that neglects the flexibility of the FG nups is incapable of explaining how the NPC can sustain a high throughput and be relatively insensitive to the removal of up to half the binding sites. Importantly, this result does not depend on the speed of the diffusion of the karyopherin–cargo complexes between the binding sites. By contrast, a model that relies on stationary binding sites predicts that transport will not be robust to deletion of half the binding sites (Figures S2 and S3). Hence, we show that not every diffusion-based mechanism can explain the robustness of transport with respect to deletion of the FG-nups. Moreover, different karyopherins can bind different specific FG-nups. Thus, they can follow different pathways within the NPC channel, each reliant on a small subset of specific FG-nups [27]; we thus predict that only removal of this small subset would prevent that karyopherin from transiting the NPC. One of the conclusions drawn by [27] from their results is that the lethal deletions have removed all the preferred FG-binding sites on an essential pathway; our model is thus completely in line with their work.
Experimental tests for our model's predictions include varying the effective potential experienced by transport factor–cargo complexes inside the NPC by systematically introducing mutations into the binding sites [16], changing the cargo size [2], or using cells with genetically modified numbers of the FG-repeat regions [27]. Finally, any device built according to the principles outlined above would possess the transport properties described by our model, suggesting strategies for the creation of highly selective artificial nanomolecular sieves.
Materials and Methods
The simulations were written in C language and run on a cluster of UNIX processors. The simulation algorithm is described in the text; see Text S2 for the actual code. Analytical calculations were in part performed with pencil and paper, or in some cases using Mathematica version 5.1.
Supporting Information
Figure S1. Several Binding Sites on a Single FG-Repeat Region Result in an Effective Potential
Figure S2. Array of Discrete Bindings Sites
Figure S3. Transport Efficiency in the Case of Stationary Binding Sites
Figure S4. Karyopherins Efficiently Exclude Nonspecifically Binding Macromolecules from the NPC, Irrespective of the Permitted Local Occupancies
Figure S5. Translocation Probability for High Local Occupancy
Text S1. Additional Information
Text S2. Simulation Code for the Diffusion of Complexes through the NPC Channel
The authors are thankful to J. Aitchison, S. Bohn, T. Chou, J. Novatt, R. Peters, S. Shvartsman, G. Stolovitzky, and B. Timney for helpful comments. This work was supported by US National Institutes of Health grants RR00862 (BTC), GM062427 (MPR), GM071329 (MPR, BTC, AZ, MOM), and RR022220 (MPR, BTC).
Author Contributions
AZ, SDT, BTC, MPR, and MOM conceived and designed the experiments and formulated the model. AZ and SDT performed the calculations and simulations. AZ, BTC, MPR, and MOM wrote the paper.
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Admittedly, I'm not a MacGyver by any measure.
That's why I was utterly relieved at the simplicity of assembling this contraption.
All I had to do was tighten a couple of screws at the base, and voila!
I was done.
Surprisingly, the post looks really good. It easily blends in with the furniture and seems to have some pretty decent workmanship.
You'll have to cough up a bit more for it, but every penny is worth it considering the aesthetics.
Now, I have a monster of a cat.
At 19 pounds, it easily knocks anything in its path. That was one of my hugest concerns while getting this post.
I wasn't sure if it would withstand the affronts of my mini-godzilla. However, I'm pleased to report that the scratcher is still standing! The base is large and steady enough to handle hours of climbing, scratching and boxing.
This particular model is also pretty tall. At 32" high, it's much larger than your ordinary cat toy.
Nevertheless, this works to the advantage of the kitties.
They can easily stretch through the whole length of their body, which is essential in toning their muscles. The height also makes for thrilling scaling adventures, keeping the playful ones entertained without end.
The durability of this SmartCat scratcher is worthy of note.
In the past, I've owned… No wait, my cat has owned carpet-based scratchers that wear out in no time. This one, however, still looks new many months after I got it. This has to do with the sisal fiber that it's made of.
My kitty seems to have tons of fun digging-in its claws, but at the end of it, the fiber still looks pristine.
The bad sides to this cat scratch post
Just like the Furminator, it took a while before my kitty took to the scratcher.
After excitedly assembling it, I waited for a reaction, some emotion even, and nothing.
She just went on with her business of ripping up the couch and looking extra cute at that. I tried moving it near the couch, as the makers suggest, and nothing.
In frustration, nay, in desperation, I sprayed a bit of catnip on it. And the rest, as they say, is history.
Despite the initial hesitation, my cat now can't get enough of this scratcher.
My couch has now been spared (Thank God!), and she is as happy as ever. For those who want to spoil their cats, a padded perch, that is sold separately, can be placed atop the post to provide a comfortable lounge for the kitty.
Should you get this scratching post?
Damn right you should!
All things considered, the SmartCat Scratcher is worth every dime. This is probably the best cat scratching post I've ever used.
So if you're looking for cat scratching posts, I highly [easyazon_link asin="B000634MH8″ locale="US" new_window="default" tag="weedesgri-20″ add_to_cart="default" cloaking="default" localization="default" nofollow="default" popups="default"]recommend you pick this up[/easyazon_link].


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Soft Updo Hair For 2010
The Trendy Look For Updos This Spring And Summer Is Soft And Romantic
Here is how to get the new softer look.
Start by rubbing a mattifying powder well into your roots for volume.
Next gently pull through your hair to the ends.
Create soft loose waves using your flat iron or curling iron.
Pin up your hair section by section to get these soft romantic looks.
Leave pieces free for effect, you can also experiment by leaving different pieces of your updo free.
For example try:
Leaving a ½ section of hair around your complete hairline free from the updo
Pin remaining hair up section by section
Next take sections of the free hair and crisscross it in a basket weave style on top of your updo pinning into place.
Enjoy, soft updo hair
© 2005-2017 Beauty And The Bath.com
All Rights Reserved World Wide


text:
Advance 9934; ca. 1961; Misses Suit and Blouse. Versatile 3-piece suit with slim skirt, jacket and sleeveless blouse.
Also released as Advance American Pattern P290.
Links to reviews/blog posts Edit
Sources/Vendors Edit
Gallery Edit
[insert your photos of this pattern made up]
Wishlist Edit
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]


text:
Welcome to Oak Park Locksmith IL
Oak Park Locksmith Oak Park Loscsmith understands your commercial locksmith needs and so Oak Park Loscsmith provides quality and effective services for competitive prices. No matter the locking problem you are dealing with, Oak Park Loscsmith is the right locksmith company in Oak Park area; so, contact Oak Park Loscsmith now. Here at Oak Park Loscsmith, our highly trained and experienced locksmith technicians can solve any kind of commercial locking problem. Oak Park Loscsmith likes to establish long term partnerships with all of our clients at Oak Park Loscsmith. Here at Oak Park Loscsmith, we are available for any of your commercial locksmith needs and Oak Park Loscsmith ensures you that Oak Park Loscsmith is the right choice to make. We, Oak Park Loscsmith are always exceeding our clients expectations as Oak Park Loscsmith is very involved in what Oak Park Loscsmith offers.
Oak Park Locksmith is a world class locksmith company operating across Oak Park. Mclaughlin's not only designs several types of locks for safes or doors but also provide repairing services timely. You can contact us any hours of day or night, if you find trouble in your locks. Technicians at Mclaughlin's are expert enough to unlock any type of locks. You can even avail installing services from Mclaughlin's . We are the master in lock and safe designing. Whether it is for digital commercial safe or residential safe, you can contact Our Company's office at Mclaughlin's are open all around the clock.
Oak Park Locksmith Oak Park Locksmiths Locksmith Oak Park Locksmiths Oak Park Locksmith Oak Park IL


text:
Yanni - The Storm 2004 Live Video HD MP3
Yanni - The Storm (Antonio Vivaldi) "The Concert Event" Live Concert from Mandalay Bay Events Center, Las Vegas 2004 Piano, Keyboards - Yanni GREECE ...
Yanni - The Storm MP3
This song was based on Vivaldi's composition, Summer (Four Seasons). Violin: Samvel Yervinyan [Armenia]; Violin: Sayaka Katsuki [Japan]; Harp: Victor ...
Yanni - The Storm (HD) MP3
Yanni On HD Thanks for all of U Band Charlie Adams (USA) -- drums Victor Espinola (Paraguay) -- harp, vocals Pedro Eustache (Venezuela) -- flute, saxophone ...
المخرج ايمن تيمور حصريا يانى Yanni - Concert 2006 MP3
المخرج ايمن تيمور https://www.youtube.com/user/aymanemk.
Yanni The Storm MP3
Yanni https://www.youtube.com/user/aymanemk.
Yanni, Violin vs Saxophone HD MP3
An amazing competition between experienced musicians.
Yanni - The Rain Must Fall MP3
Yanni's official live music video for 'The Rain Must Fall'. Click to listen to Yanni on Spotify: http://smarturl.it/YanniSpotify?IQid=YanniRMF As featured on The ...
The Rain Must Fall
YanniTe fuiste Sabiendo que yo estoy Aun amandote Llorandote Adentro Reviento por la pena Y la desolacion Sin resignacion Al beso d[...]
Yanni - World Dance (HD) MP3
Yanni On HD Thanks for all of U Band Charlie Adams (USA) -- drums Victor Espinola (Paraguay) -- harp, vocals Pedro Eustache (Venezuela) -- flute, saxophone ...
Yanni - Rainmaker MP3
Didgeridoo: David Hudson [Australia]; Violin: Samvel Yervinyan [Armenia]; Vocal: Michelle Amato [USA]; Vocal/ Harp: Victor Espinola [Paraguay] From Yanni ...
Yanni live - The storm ( Sayaka Katsuki, Samvel Yervinyan) MP3
Yanni - The Storm MP3
Yanni the storm, i like this song! The following music and content was not made by me and credit goes only to its creator(s).
Yanni - The Storm (Royal Albert Hall) MP3
Concierto de Yanni en el Royal Albert Hall de Londres el 23 de abril de 2014.
Yanni - The Storm (Live at Warsaw, Poland in 2014) MP3
Live performance of Yanni captured in the concert at Congress Hall, Warsaw on 5th May 2004.
The Storm Yanni cover by the Harmony Guys - Intro (hungarian dance ) MP3
Special Thanks to Mr. Ayman Mostafa For Rearranging "The Storm - Yanni ". The Harmoy Guys facebook : https://www.facebook.com/TheHarmonyGuys.
Yanni takes Memphis and His fans by storm! [All Access: Season 3, Episode 1] MP3
Download Yanni's collection on ITunes: http://smarturl.it/yanni Watch as Yanni performs in Memphis, serves as the resident "Duck Master" for the day and ...
Yanni-The Dream Concert In Egypt/The Storm MP3
Yanni in Egypt at the great pyramids,,The dream concert which is completely different than any concert else.The Storm #TheDreamConcert #Egypt #Yanni ...
The Storm - Yanni at Royal Albert Hall MP3
Royal Albert Hall. Londres, 23 de abril de 2014.
The Storm (Yanni) - Guitar Version MP3
Arranjo de guitarra para a música The Storm - Yanni Guitar arrangement for the song The Storm - Yanni The final version of this video: ...


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The Civil War: Chickamauga to Charleston
Chickamauga to Charleston
From 1860-1865 the United States of America was locked in a bloody conflict between Northern and Southern states that would determine the future of the Union. Join UW-Madison for the last chapter of our Civil War trilogy, covering the war's final two years in the South, but ending where it all began: at Fort Sumter in Charleston Harbor.
At a glance
What: The Civil War: Chickamauga to Charleston
When: Apr 1-9, 2017
Cost: $2,899
Tour Leader: Doug Whittle
For additional information, contact: Doug Whittle» 608-263-7787 or Kim Seymour» 608-262-3731.
What you'll experience
This will be another extraordinary educational journey, beginning in Chattanooga, Tenn., and concluding in Charleston, S.C., visiting the battlefields of Chickamauga, Lookout Mountain, and Missionary Ridge in Chattanooga, followed by visits to Kennesaw Mountain, Dunlap Hill, Walnut Creek, and Fort Sumter. Additional stops at the National Prisoner of War Museum in Andersonville, Ga., site of the infamous Confederate prison camp, Fort Jackson in Savannah, Ga., and Boone Hall Plantation near Charleston will provide background and insights into the war behind and beyond the battlefields as well. You will be joined by expert guides and historians who will bring these events to life as you walk the grounds and experience firsthand the ebb and flow of the battles and sieges that were waged between the Union and the Confederacy.
Your tour will begin in Chattanooga, with a ride on the Tennessee River aboard the River Gorge Explorer to get a sense of what an assault on Lookout Mountain entailed, and then follow that up with tours of the battlefields of Chickamauga, Lookout Mountain, and Missionary Ridge. From there you will head south toward Macon, Ga., stopping at Kennesaw Mountain, Dunlap Hill, and Walnut Creek; small battles that help make sense of the big picture. From there a stop at Andersonville provides a sense of the abject horror of war beyond the battlefield.
Concluding our journey on the Atlantic coast, we tour the beautiful downtown area of Savannah; walk the grounds and the living history exhibits at Boone Hall Plantation; see the H.L. Hunley, the first submarine to sink a warship in battle before disappearing itself for more than a hundred years; and, of course, ride a ferry to Fort Sumter to see where the whole thing began on April 12, 1861.
This is going to be another fascinating learning experience on some of the most storied ground in the United States. Expert guides, historical reenactors, fine Southern cookin', and friendly companionship; this is American history at its finest and most engaging.
You don't want to miss this one!
April 1
- Fly to Chattanooga, Tenn.
- Afternoon guided boat tour on the Tennessee River Gorge Explorer
- Dinner and overnight in Chattanooga
April 2
- Guided tour of the Battle of Chickamauga
- Dinner and overnight in Chattanooga
April 3
- Guided tours of the Battle of Lookout Mountain and the Battle of Missionary Ridge
- Dinner and overnight in Chattanooga
April 4
- Guided tour of the Battle of Kennesaw Mountain
- Depart for Macon, Ga., with tours of the Battle of Dunlap Hill
- The Battle of Walnut Creek, and Ocmulgee National Monument en route
- Dinner and overnight in Macon
April 5
- Andersonville National Historic Site
- Camp Sumter Military Prison
- National Prisoner of War Museum
- Depart for Savannah, Ga.
- Fort Jackson
- Dinner and overnight in Savannah
April 6
- Guided morning tour of Savannah
- A free afternoon to explore Savannah
- Dinner and overnight in Savannah
April 7
- Depart for Charleston, S.C.
- Fort Sumter
- Boone Hall Plantation
- Dinner and overnight in Charleston
April 8
- Charleston
- H.L. Hunley
- Civil War Walking tour
- Dinner and overnight in Charleston
April 9
- Depart for home
General Information: Everyone is welcome to join us.
You will enjoy the company of expert Civil War historians as guides, and we provide comfortable, private transportation throughout the trip. Please understand that walking is necessary in order to properly visit these historical sites.
Price-$2,899/person based upon double occupancy, plus air. This price includes all lodging, all transportation, all entry fees, all guides, daily breakfasts, all dinners, and all university staff services. Single supplement is $577.
Airfare is not included in this price. Upon receipt of your deposit our travel agent will be available to assist you with your flight arrangements if you wish, regardless of your point of origin. If you plan to make your own air arrangements, please contact us beforehand to confirm available space on the trip. Thank you.
Chattanooga, Tenn.: Courtyard by Marriott Downtown
Macon, Ga.: Marriott Macon City Centerampton Inn
Savannah, Ga.: Country Inn and Suites
Charleston, S.C.: DoubleTree by Hilton
Questions about this trip or other upcoming Educational Travel destinations? Contact Doug Whittle, 608-263-7787, email@example.com, or Kim Seymour, 608-262-3731, firstname.lastname@example.com.
Registration: To register, complete the registration form and mail it to:
Kim Seymour
Educational Travel Program
21 N Park St., 7th Floor
Madison, WI 53715


text:
An 18-year-old tool decided he'd seek his moment of infamy attempting to shoot up a community college classroom housed in a mall. He confessed to his plans to an online message board moments before he opened fire:
Three minutes before the statement was posted on online messaging board 4chan.
It said: 'Hey, it's time. My name is Neil Macinnis and I go to New River Community College in Christiansburg, Virginia, ten minutes away from Virginia tech. I'm gonna give y'all the details because the news never gets it right.
'Stevens 320 shotgun. Buck shots and slugs.'
'I'm a bit nervous because I've never really handled a shotgun but a few times with the Christiansburg police.
'Anyways this is not a highscores game but actually a lesson (that's why I'm at school)…Wish me luck. An heroing [sic] is not necessary unless I get f***ed out the ass. It's pretty busy.'
The term 'heroing' is believed to refer to committing suicide.
We all have fun mocking mall security thanks the the legend of Gecko45, but it was an off-duty mall security officer who took this turd down and keep him down until the police arrived.
There is no word on the condition of the two women injured at the college. Please consider saying a prayer for them.


text:
Butterick 5960; ca. early 1970s; Misses' Skirt, Pants and Jacket. Side wrap skirt, in two lengths and straight-legged pants are darted into waistbands. Fitted double-breasted jacket with large notched lapel collar has high center back vent, full length sleeves and flap and button trim. With or without top-stitch trim. Purchased tops.
Links to reviews/blog postsEdit
- The Vintage Cat
- [1] GoofingOff Bust 32.5
Add a photo to the gallery by clicking the "modify" button below.
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]


text:
120 Mayoral Drive
WY Building, Ground floor
Auckland City
New Zealand
Phone: +64 9 921 9637
Monday – Friday, 10am – 4pm
International Centre
AUT University
Private Bag 92006
Auckland 1142
New Zealand
AUT has a network of agents around the world who can provide information about AUT programmes and New Zealand Government regulations.
Contact an international agent
Contact the University Postgraduate Centre
Email firstname.lastname@example.com
Last updated: 02-Dec-2016 11.33am
The information on this page was correct at time of publication. For a comprehensive overview of AUT qualifications, please refer to the Academic Calendar.


text:
North Carolina (5-0) vs. Oklahoma State (4-0)
Lahaina, Maui, Hawai'i – Lahaina Civic Center
Tuesday, 5.30 p.m. (10:30 p.m. EST)
ESPN (Dan Shulman, Jay Bilas)
"Giving up 90 points still is not what we're necessarily about. We have to finish and learn how to close the game. But in a tournament a win is a win, so we survive and advance." – Oklahoma State guard Phil Forte after Monday's 98-90 win over UConn
"We've got a big-time challenge. I told them it's the big leagues tomorrow because Oklahoma State was really, really impressive tonight." – UNC head coach Roy Williams
"I think we have good players who are really good people who are trying to work really hard. I worry about all the predictions and the preseason stuff so little that I didn't even know we were picked seventh. It's not something I pay attention to, to be quite honest. We try to get better every day. We beat a very good UConn team today." – Oklahoma State head coach Brad Underwood
UNC Bounces Back: After a sluggish start to this Hawaiian road trip in Honolulu on Friday, the Tar Heels played with more defensive intensity and executed better on the offensive end in rolling past Chaminade on Monday. UNC's post trio of Isaiah Hicks, Kennedy Meeks and Tony Bradley combined for 56 points on 20-of-26 shooting as the Tar Heels shot a season-high 57.8 percent from the floor. UNC is shooting 51.2 percent on the season, while holding its opponents to 37.0 percent. The Tar Heels are also averaging 94.4 points per game, which is good sign considering they've won 50 straight when crossing the 100-point threshold.
Cowboys Spotlight: First-year head coach Brad Underwood has Oklahoma State rolling through four games. The former Stephen F. Austin head coach's squad is averaging 104.8 points per game on 50.8 percent shooting, including a 41.0 percent effort from long range. OSU is currently ninth nationally in adjusted tempo, according to kenpom.com. The Cowboys have also been effective on the glass, possessing a plus-10.2 rebounding margin. Sophomore guard Juwan Evans is the headliner, an aggressive player who is averaging 26.3 points per game on 53.1 percent shooting.
Threes Dropping: Seven different Cowboys have attempted at least eight 3-pointers through four games with plenty of success. Phil Forte is averaging three made 3-pointers per game and is connecting at a 42.9 percent clip, while Evans and Jeffrey Carroll have been quite efficient, combining to knock down 63.2 percent of their treys (12-of-19). Six players have made at least four 3-pointers thus far, thereby placing an emphasis on UNC's ability to battle around screens and contest perimeter shots from most everyone in a Cowboy jersey. Defending the 3-ball has been a point of contention for Underwood's squad as well: OSU's opponents are shooting 44.4 percent from long range.
Matchup to Watch: While Evans has earned the headlines, he's received plenty of perimeter help from Forte (21.0 ppg on 53.3 percent) and Carroll (18.5 ppg on 66.7 percent). Such high volume scoring on efficient shooting forces opposing defenses to extend beyond their comfort zones, which then stresses communication for help purposes. Roy Williams has praised the defensive efforts of Joel Berry, Kenny Williams and Nate Britt through the first two weeks of the season. They will encounter their toughest assignment to date against the Cowboys' guard-dominant approach.
Last Meeting: OSU won the only game in this series on March 26, 1946, when the school went by Oklahoma A&M. The Cowboys defeated UNC, 43-40, in the NCAA championship game at Madison Square Garden.
Projected UNC Starters:
2 Joel Berry (6-0, 195, Jr.) – 14.8 ppg, 4.6 apg
3 Kennedy Meeks (6-10, 260, Sr.) - 14.2 ppg, 10.2 rpg
4 Isaiah Hicks (6-9, 242, Sr.) - 15.0 ppg, 5.0 rpg
44 Justin Jackson (6-8, 210, Jr.) – 14.8 ppg, 3.8 rpg
24 Kenny Williams (6-4, 180, So.) - 6.0 ppg, 4.6 rpg
Projected Oklahoma State Starters:
23 Leyton Hammonds (6-8, 215, Sr.) – 7.5 ppg, 6.8 rpg
41 Mitchell Solomon (6-9, 245, Jr.) – 2.3 ppg, 3.3 rpg
01 Juwan Evans (6-1, 185, So.) - 26.3 ppg, 6.3 apg
13 Phil Forte (5-11, 195, Sr.) – 21.0 ppg, 2.8 rpg
21 Lindy Waters (6-6, 200, Fr.) – 6.3 ppg, 2.3 rpg
UNC Info
Roster & Bios
Oklahoma State Info
Roster & Bios


text: NATURE'S BEST™ The NATURE'S BEST™ concept began with simple yet dynamic goals: to celebrate the beauty and diversity of nature through the art of photography, and to use this far-reaching medium as a creative tool for encouraging greater public interest in outdoor enjoyment and conservation stewardship. The annual Windland Smith Rice International Awards program evolved from this ambitious mission to become one of the most highly-respected and visually compelling nature photography competitions in the world. More than 20,000 entries are received and judged each year in the Awards competition. Nature's Best Photography Windland Smith Rice International Awards Competition The competition opens each January and the images are judged by a panel of experts in the fields of photography, science, and conservation. Photographers whose images move into the second round of judging are emailed and we request high-resolution images, RAW files, and detailed captions. The winning images are published in the Special Collectors Editions of Nature's Best Photography. In addition, the top Winners are displayed as large-format prints in the annual exhibition at the Smithsonian's National Museum of Natural History in Washington, DC. Each photograph is judged on technical quality, originality, and artistic merit. Enter up to 25 images for the entry fee of $30 US dollars (no foreign checks). Email: firstname.lastname@example.com Nature's Best Photography Smithsonian Exhibition Open through SEPT. 2017 In partnership with the Smithsonian's National Museum of Natural History in Washington, DC, the Winners of each photographic category and a selection of the Highly Honored images from the Windland Smith Rice International Awards are displayed to millions of visitors each year. The print exhibition is accompanied by an HD video on large-screen TVs. View the awards videos below from past exhibitions on the NBP YouTube channel (set for best quality). Awards Special Publications The complete collection of finalists from each year's Windland Smith Rice International Awards is published in the Fall/Winter Special Collectors' Editions. Go to the Publications section of this website to learn more about our magazines. Subscribe to get the semi-annual delivered to your home or get the past winners in single and back issues.


text:
Naughty Lesbian NursesFavorites [+]
Pay-Per-Minute Viewing
You can now enjoy this movie on your iPhone®, iPad®, or compatible Smart TV & Android devices.
Purchase Options
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Available MP4 sizes:SD 480p (1.77 GB)
DRM-free downloads offer content that is not limited by standard Digital Rights Management (DRM) access technologies. These technologies allow hardware manufacturers, publishers, copyright holders and individuals to limit the usage of digital content.
When you purchase DRM-free downloadable content, that content is available for you to download on any computer and save to a disk whenever you like. You can also download to multiple computers. There is no timeframe associated with your purchase and the content will not expire.
Running Time: 115 minutes
Released: 12/2007
Studio Name: Doghouse Digital Mile High Media
Series: Naughty Lesbian Nurses
Director: Rod Vicious
Stars: Juicy Pearl Lexxy Lilly Sky Pamela Kayne Sophie Kream ...More
Description: Welcome to the hospital fantasy of your dreams. Lay back and enjoy the hospital stay of a lifetime as these lesbo nurses get it on with one another. These horny nurses love providing that extra care. It gets them so excited, they just have to cum. Now that's a health plan!
5 Scenes from Naughty Lesbian Nurses
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Sex Acts: Toy Play - Vaginal, Toy Play - Oral, Vaginal Sex, Oral Sex, Masturbation
Positions: Standing Missionary, Kneeling, Sitting, Standing Doggy Style, Laying, Standing
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text:
Do what thou wilt shall be the whole of the Law. Since Jonathan Korman wrote about consent, I've been giving it some deep thought. Including the comment by Niki Whiting on how women need to be educated about consent. And I've been thinking of it in context of the freak out in the atheist community [Read More…]
Violence, Children and Women
There seems to be a theme of violence in the news this morning, and before I attempt to wrap my head around the concept of idol-worship (stay tuned!) I want to talk about violence. The first regards a local story on WSB of an investigation into child abuse because of a Santeria ritual. A daycare [Read More…]
Request for Support on Georgia Harassment Story
This has been going on in my "backyard" and has been covered by The Wild Hunt already. Even with a thriving Jewish, Hindu, Muslim and Pagan population in the Atlanta area, this is still a part of the country where being religious means you're a Protestant Christian. I have to say I'm impressed by the [Read More…]


text:
Relax. You're not having a transition. Retro video activity systems have ready-made a monumental revivification in recent years, likely due to the reality that whichever kids never burgeon up. There seems to be a unadulterated tenderness among the techno-geeks out at hand for the innovative gaming systems. A casual scrabble of the computer network will yield hundreds of results for websites dyed-in-the-wool to retro gambling. These systems were all the viciousness rear in the day, and it appears that their instance has move over again.
Back once I was a schoolgirlish boy, these vice systems were like-minded a contribution from the vault of heaven. The lone way I was able to play visual communication games was to go downtown to the portico. This posed a momentous problem, as my parents were not too sharp on the mental object of rental me go downtown by myself. Furthermore, the district passageway tended to draw few untrusty characters. I skipped arts school one day and went downhill to comedy Ms. Pac-Man, but I got caught by a malingerer officer. After that incident, my parents were lonesome too zealous to buy me the new Atari.
Man, I loved that arrangement. The Atari 2600 had all the best ever games. Asteroids was, by far, my popular hobby. Dude, I could play that unfit indefinitely. Or so it seemed. What roughly speaking Pitfall? I favored that game, too. Then, Kung-Fu Master came out! I don't presume I vanished my sleeping room for a period. At the time, I swore I wouldn't physiological state until I reclaimed the event Princess Victoria. Damn those wound throwers! To this day, my parents fixed blasted that team game for the decrement in my grades that school term.
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By the time the territory picture winter sport souk round-bottomed out in 1984, I had concentrated a fairly substantial assemblage of games. I category of squandered flavour in video games say that juncture. I finished up bighearted my Atari and all of the games to my kid brother. I didn't dance it overmuch anymore, and I was too engaged annoying to get a girlfriend. In retrospect, I decision I had unbroken that system. I would worship to devote an eventide playing Space Invaders! Maybe I'll see if that row of pillars is still break open. I sensation if girls ever go here......


text:
Shallow Bay Beach - Parks Canada Gros Morne National Park
The water is shallow and warms up to swimming temperature in the summer. The northern more remote part of the beach is backed by high dunes, and can receive impressive waves after a storm. The area attracts shorebirds, especially in the fall, there can be hundreds of migrating shorebirds clustered on the upper beach at high tide.
Location data provided by the operator. Please confirm location before departure.
Unsupervised beach, large gravelled parking area located 100m from the beach, picnic shelter with wood stove, flush toilets, drinking water, changing rooms, picnic tables, playground, 1km trail, viewing platform with benches, interpretive signs, campground nearby.
Helpful Information
Contact Information
Driving distances and calculations derived using Google Maps. Actual driving times may vary. GPS coordinates have been provided by tourism operators. Please confirm location with operator before departure.
Special Offers from this Operator
- Lobster Cove Head Lighthouse, Gros Morne National Park
- +1 (709) 458 2417
- firstname.lastname@example.com
- Broom Point
- (709) 458 2417
- email@example.com
* Offers are subject to availability. Taxes are extra. You may have to pre-book and/or mention
the promotion at the time of booking. Please check with the operator for more details.