Thou Shalt Not Electioneer: Religious Nonprofit Political Activity and the Threat “God PACs” Pose to Democracy and Religion

The Supreme Court’s 2010 decision in Citizens United v. FEC invalidated a longstanding restriction on corporate and union campaign spending in federal elections, freeing entities with diverse political goals to spend unlimited amounts supporting candidates for federal office. Houses of worship and other religious nonprofits, however, remain strictly prohibited from engaging in partisan political activity as a condition of tax-exempt status under Internal Revenue Code § 501(c)(3). Absent this “electioneering prohibition,” religious nonprofits would be very attractive vehicles for political activity. These 501(c)(3) organizations can attract donors with the incentive of tax deductions for contributions. Moreover, houses of worship need not file with a government agency to begin operating and deriving tax benefits, and the IRS has shown reluctance to aggressively audit their activities. Two circuits have previously upheld the electioneering prohibition against legal challenges, but recent jurisprudential shifts expose the tax code provision to challenge under the Religious Freedom Restoration Act (RFRA), which directs courts to apply strict scrutiny to facially neutral laws that substantially burden the free exercise of religion. First, Burwell v. Hobby Lobby Stores, Inc. greatly reduced the barriers to successful RFRA claims. Second, by lifting restrictions on political speech for many other types of organizations, Citizens United magnified the burden the electioneering prohibition imposes on religious organizations. The decision also rejected compelling state interests that might have previously shielded the law from invalidation. This Note is the first analysis of the electioneering prohibition’s vulnerability in this new legal climate. Despite these significant developments, this Note ultimately concludes that the electioneering prohibition can survive RFRA challenges because the prospect for widespread use of religious organizations as conduits for political activity undermines the values reflected in Establishment Clause jurisprudence

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3′(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089–1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI(3)P, was differentially regulating endocytosis from the two domains, we examined the PI(3)P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI(3)P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI(3)P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI(3)P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition

Local signaling by the EGF receptor

Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling

The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila

Degradation of cytoplasmic components by autophagy requires the class III phosphatidylinositol 3 (PI(3))–kinase Vps34, but the mechanisms by which this kinase and its lipid product PI(3) phosphate (PI(3)P) promote autophagy are unclear. In mammalian cells, Vps34, with the proautophagic tumor suppressors Beclin1/Atg6, Bif-1, and UVRAG, forms a multiprotein complex that initiates autophagosome formation. Distinct Vps34 complexes also regulate endocytic processes that are critical for late-stage autophagosome-lysosome fusion. In contrast, Vps34 may also transduce activating nutrient signals to mammalian target of rapamycin (TOR), a negative regulator of autophagy. To determine potential in vivo functions of Vps34, we generated mutations in the single Drosophila melanogaster Vps34 orthologue, causing cell-autonomous disruption of autophagosome/autolysosome formation in larval fat body cells. Endocytosis is also disrupted in Vps34−/− animals, but we demonstrate that this does not account for their autophagy defect. Unexpectedly, TOR signaling is unaffected in Vps34 mutants, indicating that Vps34 does not act upstream of TOR in this system. Instead, we show that TOR/Atg1 signaling regulates the starvation-induced recruitment of PI(3)P to nascent autophagosomes. Our results suggest that Vps34 is regulated by TOR-dependent nutrient signals directly at sites of autophagosome formation

WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous–related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity