After washing twice in PBS-Tween 0.1%, sections were incubated (O/N; 4°C) with primary antibodies diluted in a fish gelatin blocking solution of PBS1x (pH 7.4), 0.5% Tween, 10% glycerol (v/v), 18% D(+)-Glucose (w/v), and 4.5% fish skin gelatin (G-7765; Sigma). DAB staining was performed using a Vectastain ABC kit (Vector Labs) and Peroxidase substrate DAB kit (Vector Labs), following the supplier’s instructions. Sections were mounted Selleckchem E7080 using VectaMount (Vector Labs). Immunofluorescence was performed using the appropriate conjugated secondary antibodies (Jackson ImmunoResearch). Sections were mounted using Fluoromont-G (SouthernBiotech). Colocalization analyses were performed using

a LSM 780 confocal microscope (Zeiss) with Zen 2011 software. Electron microscopy of retinal sections was performed as described previously

(Prasad et al., 2006). Phagosome counts were performed as described previously (Nandrot et al., 2007), using 8 μm fixed retinal sections stained with an anti-opsin antibody (see above). Sections were prepared from mice sacrificed and perfused at 6:30 a.m., 30 min after lights-on in our animal facility. Opsin-positive vesicles contained within the RPE layer (visualized at 80×) were scored for entire retinal sections, and the observer was blind to the genotype of the section. The length of the single-cell RPE layer in each section was measured using ImageJ, and the results expressed as phagosomes per 100 μm RPE length. This work was supported by grants from the National Institutes of Health (R01 AI077058 and see more R01 AI101400, to G.L.), the European Union (Marie Curie grant IRG-256319, to T. B.-C.), and the Israel Science Foundation (grant 984/12, to T. B.-C.), by the Salk Institute

(NIH Cancer Fenbendazole Center Grant CA014195), and by postdoctoral fellowships from the Leukemia and Lymphoma Society (to E.D.L.) and the Fundación Ramón Areces (to P.G.T.). “
“The family of A kinase-anchoring proteins (AKAPs) has emerged as a convergent point of diverse signals to achieve spatiotemporal specificity. Besides the extensive studies on its regulation of ion-channel activity and trafficking, AKAP79/150 (human AKAP79/rodent AKAP150) has been shown to be intimately involved in synaptic plasticity, and learning and memory (Horne and Dell’Acqua, 2007; Lu et al., 2007; Tavalin et al., 2002; Tunquist et al., 2008; Weisenhaus et al., 2010). A direct role of AKAP79/150 in gene transcription has been implicated, highlighting nuclear or plasma membrane complexes it organizes with signaling components of cAMP/CREB or calcineurin (CaN)/nuclear factor of activated T cell (NFAT) signaling pathways (Oliveria et al., 2007; Sample et al., 2012). NFAT transcription factors are activated by intracellular Ca2+ (Ca2+i) signals in concert with CaN and play critical roles in neural development, axon growth, and β-amyloid neurotoxicity (Graef et al., 1999, 2003; Hudry et al., 2012; Wu et al., 2012).

In the absence of known transcriptional determinants of proprioceptor subtype, our findings raise the possibility that certain aspects of pSN diversity are determined by graded variation in the strength of extrinsic signaling rather than

by subclass-specific expression of intrinsic transcriptional determinants. Our analysis of sensory neuron differentiation has uncovered a previously unappreciated feature of pSN diversity: the mosaic, muscle-by-muscle, dependence on Etv1 for pSN survival. The status of Etv1-sensitivity correlates inversely with muscle NT3 levels: muscles innervated by Etv1-dependent pSNs express ∼5-fold lower NT3 levels than muscles innervated by Etv1-independent pSNs. A causal role for NT3 in pSN diversification is suggested by the observation that elevation of muscle NT3 levels in Etv1−/− mice restores Etv1-sensitive pSN neuronal number, click here intraspinal sensory axonal projections, and innervation of muscle spindles. These observations extend earlier studies ( Li et al., 2006). They argue that elevated NT3 signaling activates downstream pathways similar or identical

to those activated by Etv1 itself, but does so to differing degrees depending on precise muscle target ( Figure 8A). We speculate that in addition to promoting pSN survival, graded NT3 signaling may also elicit distinct molecular responses in pSNs innervating different muscle targets, thus contributing to the functional diversity of pSNs. Indeed, changing muscle NT3 expression levels in transgenic mice has been reported to erode the selective selleck chemicals llc connectivity of proprioceptive afferents with target MNs ( Wang et al., 2007). Furthermore, recent studies have demonstrated profound changes in gene expression in pSNs in response to elevated NT3 signaling ( Lee et al., 2012). Our findings have not yet resolved whether Etv1 controls Purple acid phosphatases pSN survival through direct or indirect actions. The early loss of pSNs in Etv1 mutants could reflect a direct action of Etv1 in repressing core apoptotic programs that govern pSN survival. Because Etv1 expression in pSNs is induced by NT3 signaling ( Patel et al.,

2003), it could serve as a transcriptional intermediary in the trophic factor-mediated repression of apoptotic programs ( Figure 8B). The idea of an antiapoptotic function for Etv1, restricted to a select neuronal subtype, bears similarities to the role of the Ces-2 transcription factor in C. elegans, which engages in dedicated pathways that control apoptosis in neuronal subsets ( Metzstein et al., 1996). Moreover, in spinal neurons, the Ces2-related transcription factor E4PB4 has been shown to act in conjunction with extracellular signaling pathways to regulate the survival of MNs ( Junghans et al., 2004). Alternatively, Etv1 could control pSN survival indirectly, through regulation of other ancillary aspects of differentiation that impinge on apoptotic pathways ( Figure 8B).

Their behavior was thus similar to that of normal rats trained only up to the initial criterion for acquisition (Figure 1). On subsequent PP rewarded

days, all rats learned to avoid the devalued goal with tasting experience (Figures 8C and 8D). Thus, targeted disruption of IL activity during the overtraining period selectively prevented habit acquisition. Our findings demonstrate that both DLS-associated sensorimotor circuits and IL-associated limbic circuits register habits by heightened representations of action boundaries with diminished spike activity during decision-making periods. As the structure of these bracketing patterns click here increased with habit formation in both regions, variability in spike timing declined and single-event selectivity of individual units increased, suggesting a cross-circuit

shift from neural exploration to exploitation as behavior became automatized into a habit (Barnes et al., 2005). Despite these similarities, the IL cortex and the DLS expressed spiking changes with strikingly different temporal dynamics during learning and with different relations to the behavioral parameters being see more acquired. Even within the IL cortex, different depth levels acquired different patterns. The perturbation of IL activity that we applied by optogenetic neuromodulation during overtraining established that IL activity during this habit crystallization period is necessary for full habit acquisition. We suggest an extension of current habit learning models to incorporate dynamic neural operators in both IL cortex and DLS. By this dual-operator account, habits are composites of multiple core neural components working simultaneously, and the mark of a fully formed habit could include the alignment of task-bracketing activity Selleck Hydroxychloroquine patterns in both limbic and sensorimotor circuits. In accord with experimental evidence, associative learning models have suggested that the brain has goal-directed, action-outcome (A-O) systems comprising model-based

(e.g., tree-search) planning systems and that these compete for behavioral control with habit systems viewed as stimulus-response (S-R) or model-free systems (Balleine et al., 2009, Daw et al., 2005, Dickinson, 1985 and Killcross and Coutureau, 2003). In these frameworks, the DLS is considered to represent the core S-R association or cached model-free predictions of a habit that can be acquired early and can control behavior when selected, whereas the IL cortex serves as an executive controller or arbiter favoring habit systems (Balleine et al., 2009, Daw et al., 2005, Dickinson, 1985 and Killcross and Coutureau, 2003). The dynamics of neural activity that we observed are consistent with some predictions of these models, but there are also inconsistencies that encourage extensions of these views. At a behavioral level, we found that deliberations did not covary perfectly with outcome value expectations.

, 2011). To incorporate competitor strength-dependent

inhibition, we took the suppression factors sin and sout to be proportional to the activity I of the inhibitory units driven by stimuli located outside the Cilengitide in vitro RF: equation(4) sin=din·I,sout=dout·Isin=din·I,sout=dout·Iwhere din and dout were proportionality constants, and I was the inhibitory activity driven by the competitor. Recordings of Imc responses to single looming stimuli have shown that they are well fit by sigmoidal functions (S.M., unpublished data). Consequently, inhibitory activity as a function of the loom speed of the competitor stimulus was modeled as having the same form as Equation 1: equation(5) I=m+h(lklk+s50k) The free parameters were m, the minimum response; h, the maximum change in response; S50, the loom speed that yields a half-maximum response; and k, a factor that controls response saturation. The effect

of changing the values of each of these parameters on I is illustrated in Figure S1. A linear dependence between the input and output divisive factors (sin and sout) and the inhibitory activity (I) was assumed in Equation 4 for simplicity. This formulation minimized the number of free parameters in the model, while still allowing for nonlinear competitor strength-dependent response suppression, due to the FRAX597 datasheet nonlinearity of I. We now describe the special response properties underlying strongest versus other categorization that need to be accounted for by the model. These were revealed in experiments in which a looming stimulus of fixed speed was presented inside the RF, while a second competing stimulus of variable speed was presented far outside the RF, about 30° away. The resulting responses are referred to as the competitor strength-response profile, or CRP (Mysore et al., 2011). Essential to the explicit representation of categories in the OTid is the abrupt, switch-like increase in response suppression, observed in about 30% of OTid neurons, Carnitine dehydrogenase as the strength of a competing stimulus is increased (Figure 2D, right). The abruptness of the transition is quantified as the range of competitor strengths

over which CRP responses drop from 10% to 90% of the maximum change in response and is referred to as the transition range. Switch-like CRPs were defined as those for which the transition range was very narrow: ≤4°/s, equivalent to ≤1/5 of the full range of loom speeds tested. Population activity patterns that include switch-like responses (along with non-switch-like responses) explicitly categorize stimuli into two categories, strongest and others, as determined by crosscorrelational analysis (Mysore and Knudsen, 2011a). Conversely, excluding the top 20% of the neurons with the most abrupt response transitions (switch-like responses) from the population analysis eliminates categorization by the population activity.

In Latin America http://www.selleckchem.com/products/sorafenib.html A. cajennense is one of the main vectors of Rickettsia rickettsi, the causal agent of Rocky Mountain spotted fever ( Parola et al., 2005). This tick completes only one generation each year and shows a distinct seasonality. In Central Brazil adults predominate in the hot, rainy season

(November to March); six-legged larvae hatch in the drier and colder season (March to July) followed by the eight-legged nymphs. Both immature stages can frequently be found in pastures where they avidly attack hosts moving past the vegetation on which the ticks rest. Free-living tick stages distributed in large areas are difficult to control with synthetic acaricides, but pathogenic microorganisms, especially fungi, act as natural antagonists GPCR Compound Library ic50 of many arthropod pests and may possibly be particularly valuable for integrated tick control ( Samish et al., 2004, Fernandes and Bittencourt, 2008 and Tuininga et al., 2009). Both Beauveria bassiana and Metarhizium anisopliae can infect eggs, larvae, nymphs and adults of A. cajennense under laboratory conditions ( Lopes et al., 2007 and Fernandes and Bittencourt, 2008) but nothing is known about naturally occurring mycoses of this tick in the field. Rhipicephalus

sanguineus, another important ixodid and potential vector of R. rickettsii in the neotropics, mainly attacks dogs but can also affect humans ( Parola et al., 2005). Highly virulent fungi adapted to the target tick species and to regional climatic conditions can provide important starting points for developing effective biorational mycoacaricides. The present study reports the first isolations of pathogenic fungi from Ribose-5-phosphate isomerase field-collected A. cajennense or from their natural off-host habitats and demonstrates their pathogenicity to A. cajennense and R. sanguineus. Live A. cajennense ticks and soil samples from their habitats were collected once

a month from October 2009 to March 2011 from the privately owned Santa Branca Farm, ca. 40 km NE of Goiânia in Central Brazil (16°23′41″S; 49°04′47″W, WGS 84). A. cajennense is frequent in this area and can be found on various hosts but most prominently on horses and capybaras. Humans are also affected by this tick but human incidences of spotted fever have never been reported from the studied area. Locations where soils were collected were randomly chosen in human-made pastures (Brachiaria decumbens, Poaceae) and did not change throughout the study. These sites are protected against continuous sunlight by vegetation and are preferred resting places for horses, livestock and capybaras. From each of eight locations (all separated by at least 100 m), 25 g of mineral soil were scraped to a depth to 2–3 cm after removing leaf litter or other organic matter, transferred to a plastic bag and stored in a polystyrene cooler at 20 °C until being processed in the laboratory within a few hours of collection. On the same dates at least 100 A.

We generated long-tailed degree distributions using the power law with exponential cut-off described in Section 2, and found that the average distance to the empirical distributions was about 5.2 times zB. We then applied each of the rounding schemes described in Section 2. Scheme 1 (rounding all degrees up by 5) and Scheme 2 (by 10), reduced the factor from 5.2 to 2.7 and 2.3, respectively. Scheme 3 (adding 5 to every degree) increased the distance somewhat to 5.5. However, the more sophisticated Cobimetinib Scheme 4 (rounding to the nearest 10 for k < 100 and to the nearest 100 for k > 100)

reduced the factor to 1.4; while Scheme 5, which is like Scheme 4 but also draws all degrees under 10 from the combined Bristol distributions, decreases this factor further still to 1.2, Table S3. In other words, these schemes produce distributions almost as close to the empirical ones as the two Bristol datasets are to each other. Note, however, that the level of interference involved in Schemes 4 and 5 should be seen as the minimum reporting error required to obtain realistic reported distributions from smooth underlying ones. If in fact it were the individuals with few contacts who nonetheless claimed to have hundreds while the

highly connected reported only a small number, this would not be evident in the data. The bias introduced by inaccuracies in reported degrees which we go on to analyse should therefore be regarded as a lower bound to the potential importance of this Plasmin effect. Inaccuracy in reported degrees this website had a large effect on the reliability of estimates of prevalence and incidence (Fig. 2). The top half of Fig. 2 shows estimates of prevalence from RDS surveys where degree was mis-reported by the 5 rounding schemes. The estimates were calculated using the Volz–Heckathorn estimator. Mis-reporting degrees caused all surveys to over-estimate prevalence (compare to the ‘Actual’ prevalence in the

whole network, top). However, if degrees were correctly reported (standard RDS) the average prevalence estimate from 100 surveys was accurate, but individual variation was large. Even with inaccurate degreees, the adjusted estimates (blue bars) were still closer to the true prevalence or incidence than the point estimate from the raw data (green bars). Two of our degree-biasing rounding schemes were based on degrees collected in Bristol, UK. Scheme 4 adjusted only those degrees larger than 10: the prevalence estimate is comparable with the estimate using correct degrees. However, the error increased when inaccuracies were added to the lower degrees (1 ≤ d ≤ 10) in Scheme 5. Those with low degree have a higher weighting in the estimator (Eq. (1)) than those with high degree; therefore mis-reporting these degrees had a larger effect on the estimate. The average prevalence for rounding Scheme 5 was 39.8% [31.1–51.4% 95% CI] compared to the actual average prevalence of 27.2% [26.1–28.4% 95% CI].

, 2008, Cuijpers et al., 2010 and Cuijpers et al., 2011). Genetic analysis, by identifying risk variants and thereby increasing our understanding of how MD arises, could lead to improved prevention and the development of new and more effective therapies. Although genetic analysis has identified risk loci for many other common medical diseases (Hindorff et al., 2009), success has yet to visit MD. In this Review, we consider what has selleck kinase inhibitor so far been learnt, consider reasons for the difficulties encountered, and propose how these might be overcome. We start by reviewing evidence from the genetic epidemiology literature

relevant to the genetic basis of MD. We then consider what genome-wide association studies (GWASs) have told us. The GWAS results PLX3397 cell line are particularly important for interpreting the large, forbidding literature on candidate

gene studies, which we review next. In addition, GWAS findings inform us about the extent to which rare but more highly penetrant genetic variants might contribute to liability to MD. We finally examine whether there exist forms of MD that might be more genetically homogeneous and consider how these might be identified. Studies showing that MD aggregates within families date back to the early decades of the 20th century (reviewed in Tsuang and Faraone, 1990). Meta-analysis of the highest-quality family studies produced an estimated odds ratio for increased risk for MD in first-degree relatives of MD probands of 2.84 (Sullivan et al., 2000). Surprisingly, no high-quality adoption study of MD has been performed, so our evidence of the role of genetic factors in its etiology comes solely from twin studies. While the first of these also date to early in the 20th century, only six high-quality studies were identified in the Review completed in 2000 (Sullivan et al., 2000). Meta-analysis estimated heritability for MD to be

37% (95% confidence intervals 31–42). There was no evidence from these studies that shared environmental factors contributed meaningfully to the familial aggregation for MD. One particularly large-sample twin study of MD estimated Aciclovir the heritability of MD at 38% (Kendler et al., 2006). Epidemiological studies of MD have consistently shown a higher prevalence rate for women (Weissman et al., 1993 and Weissman et al., 1996). Therefore, twin researchers have been interested in asking whether the heritability of MD differs across sexes and, more interestingly, whether the same genetic factors impact on risk for MD in men and women. The two major studies that have addressed this question found reassuringly similar answers (Kendler et al., 2001 and Kendler et al., 2006). In both studies, MD was appreciably more heritable in women than in men (40% versus 30% and 42 versus 29%, respectively) and clear evidence was found for sex-specific genetic effects with genetic correlations estimated at +0.55 and +0.63. A substantial proportion of genetic risk factors for MD appeared to be shared in men and women.

In the dorsal vagal complex (NTS/DMV), all P-STAT3 expression was detected in non-GABAergic neurons Selleck SCR7 ( Figure 4C). When LEPRs were deleted from GABAergic neurons, all colocalization disappeared; residual

P-STAT3 was restricted to non-GABAergic neurons ( Figures 4D–4F). Thus, leptin-responsive GABAergic neurons are located in the arcuate, the DMH, and the lateral hypothalamus. With regard to glutamatergic (VGLUT2+) neurons, in control mice, P-STAT3 colocalized with GFP only in the arcuate (small number of neurons, Figure 4G), the VMH (Figure 4H), the PMv (Figure 4I), and in the NTS/DMV (Figure 4J). When LEPRs were deleted from glutamatergic neurons, colocalization disappeared in the arcuate (Figure 4K) and in the VMH, PMv, and NTS/DMV, all P-STAT3 signal was lost (Figures 4L–4N). These findings indicate Adriamycin ic50 that leptin-responsive glutamatergic neurons are located primarily in the VMH, the PMv, and the NTS/DMV (with a smaller number also found in the arcuate), and of note, in the VMH, PMv, and NTS/DMV, 100% of LEPR-expressing neurons are glutamatergic. POMC neurons play a critical role in preventing obesity as evidenced by massive weight gain in mice lacking αMSH (Smart et al., 2006 and Yaswen et al., 1999), its receptor, MC4R (Balthasar et al., 2005 and Huszar et al., 1997),

and in mice with ablation of POMC neurons (Xu et al., 2005). Given this, we examined whether POMC neurons are downstream of leptin-responsive GABAergic neurons. Specifically, we recorded inhibitory postsynaptic currents (IPSCs)

in POMC neurons (visualized with the POMC-hrGFP BAC transgene) and assessed effects of leptin. Of interest, a prior study with 200 μm thick coronal slices found that leptin reduced IPSC frequency in POMC neurons by 25% in one-third of POMC neurons and this was attributed to AgRP/NPY GABAergic neurons (Cowley et al., 2001). In our studies, we prepared thicker slices (300 μm), positing that this might preserve Electron transport chain more connections between the GABAergic and POMC neurons. In Figure 5, Figure 6 and Figure 7, we report effects on all neurons tested. Addition of leptin decreased spontaneous IPSC (sIPSC) frequency in POMC neurons by 40% (Figures 5A and 5B). This effect was not dependent upon action potentials because in the presence of tetrodotoxin (TTX) leptin reduced miniature IPSC (mIPSC) frequency to a similar extent (Figure S4A). We and others (Cowley et al., 2001 and Pinto et al., 2004) have observed that frequency and amplitude of sIPSCs in POMC neurons are minimally affected by the addition of TTX, demonstrating that most sIPSCs in POMC neurons in the context of brain slice preparations originate from spontaneous vesicle fusion events in presynaptic GABAergic neurons.

Care was taken to only evaluate retinas where the entire whole mount was obtained by dissection. Student’s t tests were used for statistical comparisons of RGC numbers between wild-type and mutant retinae. We thank Dr. Gregory Dressler for the cadherin-6 antibody and Tom Clandinin and Maureen Estevez for their helpful suggestions. This Selleckchem RG7204 work was supported by NIH R01 EY014689 (D.A.F.), NIH R01 EY07360

(S.B.), NIH EY17832 to (B.V.), NIH R21 EY018320 and NIH R01 EY11310 (B.A.B), and NIH R01 EY12793 (D.M.B.) and the E. Matilda Ziegler Foundation for the Blind (A.D.H.). “
“During the development of neural circuits, axons navigate complex cellular environments to form synapses with specific cell types and at specific subcellular locations. Consequently, a neuron that receives synaptic input from multiple presynaptic sources will often develop distinct types of synapses unique to each input. Although progress has been made in understanding general mechanisms of axon guidance and synaptogenesis,

the molecular mechanisms that regulate the formation and differentiation of specific classes of synapses in the mammalian central nervous system are poorly understood. The hippocampus is an excellent model for studying the development of specific classes of synapses because the pattern of connectivity between different cell types is well characterized, and different classes of synapses are structurally distinct (Figures 1A–1D). This is most strikingly exemplified by mossy fiber synapses that connect dentate gyrus (DG) and CA3 neurons. The mossy fiber presynaptic terminal consists of a large and complex Caspase inhibitor presynaptic bouton that grows 50–100 times larger in volume than a typical asymmetric synapse and can contain over 30 separate vesicle release sites (Chicurel

and Harris, 1992 and Rollenhagen et al., 2007). The postsynaptic structure on the CA3 dendrite consists of an equally elaborate multiheaded spine known as a thorny excrescence (TE) (Figure 1D) (Amaral DOK2 and Dent, 1981). Because of its enormous size and position near the soma of CA3 neurons, activation of a single mossy fiber synapse can cause the CA3 neuron to fire and, therefore, has been called a “detonator” synapse (McNaughton and Morris, 1987). Farther from the soma, CA3 neurons also receive synaptic input from other CA3 neurons and the entorhinal cortex onto typical asymmetric synapses (Figures 1B and 1C). The molecular mechanisms that drive initial formation and maturation of these unique hippocampal mossy fiber synapses remain unknown and are likely to be distinct from those signals that govern typical asymmetric synapse formation. Evidence in support for a role of molecular interactions in regulating the differentiation of specific classes of synapses comes largely from genetic studies in invertebrates (Ackley and Jin, 2004 and Rose and Chiba, 2000). For example, in C.

Even in ideal situations, the optical detection of the membrane potential can only be carried out with relatively few emitted photons. Because of this, for the signal to be distinct from the photon shot noise, one typically needs to use very efficient chromophores, very strong light sources, or extensive AZD6244 manufacturer temporal or spatial averaging. Unfortunately, despite its great strength as an insulating layer and in maintaining cellular integrity, the plasma membrane is also a very delicate part of the cell and does not tolerate intense illumination. The photodamage associated with excited state reactions,

such as the generation of disruptive oxygen free radicals and other triplet state reactions, or simply by local heating, following photoabsorption by the chromophores used to measure the voltage signals, can easily

compromise the integrity of the membrane and kill the cell. Indeed, some sort of photodamage is present in essentially all voltage imaging measurements and is GSK1120212 clinical trial normally the reason voltage imaging experiments are terminated. To make this situation worse, neurons, like most mammalian cells, have a significant complement of endogenous chromophores, such as flavins, cryptochromes, and phorphyrins, that absorb visible light and, in some cases, are even located near the membrane. So even illuminating unstained neurons can lead to the generation of oxygen free radicals, damaging the membrane and altering membrane conductances, and may even result in membrane perforations ( Hirase et al., 2002). This endogenous photodamage is so prevalent that one sometimes wonders whether neurons have light-sensing machinery, as unicellular organisms do, to monitor circadian light changes. A third constraint arises from the fact that most of the membranes in cells are actually internal membranes. The plasma membrane, the only one across which the neuronal

membrane potential exists, is only a small proportion of the total membrane surface in the neuron. Thus, any chromophore that binds indiscriminately to membranes will mostly bind to internal membranes, which have no direct sensitivity to the plasma membrane voltage, and as a result, these and chromophores will merely contribute to the background noise of the measurement. This is quite a significant problem, one that again does not exist for calcium imaging, where the intracellular calcium eventually equilibrates by diffusion in the cytoplasm, in principle making every molecule of chromophore in the cytosol a possible contributor to measuring the signal. For voltage imaging, the desire to target only the plasma membrane and yet avoid internal membranes compounds the already strong localization requirements.