The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2–mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.

Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H42RA) exhibited a significant loss of global H2Bub1. H42RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter, to control the subcellular location of the H2Bub1 E3 ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H42RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H42RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications.

With issues of immigration policy very much at the forefront of the 2016 elections, this annual conference offers policy and legal analysis on key immigration topics facing the country and the incoming administration and Congress in 2017.

The 13th annual Immigration Law & Policy Conference offered policy and legal analysis on key immigration topics, including: the election and the future of immigration policy; refugee resettlement in the United States; immigration detention and enforcement; and the impacts of the Supreme Court opinion in the important U.S. vs Texas case

A discussion featuring data on immigration trends and the agricultural workforce, and some of the adjustments that farm employers are making, including increased mechanization, improved wages and benefits, and the increased use of the H-2A program. University of California-Davis’s Phil Martin, along with researchers from the the U.S. Department of Agriculture, U.S. Department of Labor, present their findings on the foreign agricultural workforce in the United States, which is followed by comments from the President of Farmworker Justice on some of the policy implications.

Amid ongoing Brexit negotiations, much remains uncertain for the roughly 1 million UK citizens living elsewhere in the European Union. This report offers a demographic profile of these Brexpats, considering what form an EU-UK deal on citizens’ rights might take and identifying key challenges many Britons are likely to face—including difficulty securing legal status and accessing labor markets, social security, and health-care systems.

As policymakers in a number of countries, the United States among them, debate limiting family-based immigration, this issue brief explores family-migration trends and policies in the United States, Australia, Canada, the United Kingdom, and several other European countries. Family admissions play a key role, even in countries that prioritize economic or other immigration streams.

Who is an immigrant? Does that status change if, for example, a foreigner marries a native-born resident or serves in his or her adopted country's military? This explainer answers basic questions about international migrants—who they are, their top destinations, where they come from, how they are counted, and more.

With thousands more migrants potentially traveling through the Western Balkans this year, this MPI Europe webinar explores the implications of the buttressed EU border on the bloc’s neighbors, including the issues of outsourcing migration control, EU support for addressing irregular migration in neighboring countries, and considerations for EU policymakers.

With thousands migrants potentially traveling through the Western Balkans this year, this MPI Europe webinar explores the implications of the buttressed EU border on the bloc’s neighbors, the migrants transiting these routes, and the local communities. Experts also explored how the European Union can support efforts to address irregular migration in neighboring countries, and what are the tradeoffs and considerations that policymakers must weigh. 

As the U.S. health-care system sags under the strain of the COVID-19 pandemic, health-care workers are not only on the frontlines fighting the virus, they are also some of the most at-risk individuals, making it more essential than ever to have sufficient numbers of qualified health professionals. While 1.5 million immigrants and refugees are already working in the U.S. health-care system, an additional 263,000 immigrants and refugees in the U.S. with health-related college degrees are working in low-skilled jobs or are out of work.

About 2.1 million immigrants work in jobs growing, harvesting, processing, and selling food in the United States, serving an essential role in feeding America. While immigrants accounted for 17 percent of all civilian employed workers in the United States between 2014-18, they played an outsized role in food production, making up 22 percent of workers in the U.S. food supply chain. They represent far larger shares in certain food-related occupations, and in particular states, as this infographic shows.

The White House immigration plan offered as a solution to resolve the fate of DREAMers seeks legal immigration cuts unlike any seen since 1924. In addition to a decrease of up to 40 percent in family-sponsored immigration, the proposal demands vast increases in enforcement and a retrenchment in protections for those seeking humanitarian relief. In exchange, one-sixth of the unauthorized population could gain legal status.

Marking the release of MPI President Andrew Selee's latest book, speakers explore emerging trends in migration, economic interdependence, technology innovation, and cultural exchange that are transforming the relationship between the United States and Mexico, and the policy implications of these changes for the future.

What President Trump calls a border crisis is in fact a crisis in the asylum system—one worsened at every turn by his administration’s harsh policies and rhetoric. Rather than spend $5.7 billion on a wall, it would be far more effective to use the money to retool an overwhelmed asylum system, adapt outmatched border enforcement infrastructure to respond to the changing composition of arrivals, and work cooperatively with Mexico to tackle the factors propelling Central Americans to flee.

Though a faceoff between the U.S. executive and legislative branches is now in the courts, with President Trump's decision to declare a national emergency so he can allocate more money for construction of a border wall, a less-noted dispute has been taking place over the Department of Homeland Security's decision to add thousands more immigration detention beds than Congress provides annually, as this article explains.

The House is set to vote on two bills that would largely dismantle the U.S. asylum system at the southern border by significantly narrowing grounds to apply for asylum, eliminating protections for the vast majority of unaccompanied minors, and unilaterally declaring Mexico a safe third country. The result would be a sharp reduction in the number of people permitted to seek humanitarian protection, as this commentary explains.

President Trump has made reshaping the U.S. immigration system a top priority. Yet the fragmented nature of policy-making in the United States—with power split between branches and levels of government—has made it difficult to pursue some of his most ambitious proposals. This report explores the evolution of migration policy under Trump, and what these changes may mean in the long run.

A report release and presentation of first-ever U.S. estimates on the actual economic costs of skill underutilization for immigrants, their families, and the U.S. economy, in terms of forgone earnings and unrealized federal, state, and local taxes.

In 2015, 43.3 million immigrants lived in the United States, comprising 13.5 percent of the population. The foreign-born population grew more slowly than in prior years, up 2 percent from 2014. Get sought-after data on U.S. immigration trends, including top countries of origin, Mexican migration, refugee admissions, illegal immigration, health-care coverage, and much more in this Spotlight article.

Immigrants from India are the second-largest foreign-born group in the United States, after Mexicans. Indian immigrants tend to be far more highly educated and have greater English proficiency than the foreign-born population overall. This Spotlight article offers the latest data on Indian immigrants, focusing on population size, state- and city-level distribution, occupation, educational attainment, and more.

The Chinese represent the third-largest immigrant population in the United States, their numbers having grown rapidly in recent decades. The population is atypical in some respects: Far more highly educated and likely to have come via student and employment pathways than the overall U.S. foreign-born population. This article offers key data on Chinese immigrants, including top destinations, incomes, and English proficiency.

The United States is by far the world's top migration destination, home to roughly one-fifth of all global migrants. In 2016, nearly 44 million immigrants lived in the United States, comprising 13.5 percent of the country's population. Get the most sought-after data available on immigrants and immigration trends, including top countries of origin, legal immigration pathways, enforcement actions, health-care coverage, and much more.

Though small, the population of Canadians in the United States is quite diverse, and includes students, highly skilled professionals on H-1B or NAFTA visas, family migrants, and retirees. Canadian immigrants have much higher educational attainment and incomes than the native- and overall foreign-born populations. This article offers an interesting data snapshot of Canadians in the United States.

Award winners were honored at the second annual awards ceremony in Washington, DC, on May 18, 2010.

Winners of the 2nd annual E Pluribus Unum Prizes awarded to exceptional immigrant integration initiatives by MPI's National Center on Immigrant Integration Policy are the Illinois New Americans Integration Initiative; the Latino Community Credit Union in Durham, NC; Tacoma Community House in Tacoma, WA; and Upwardly Global in San Francisco, CA. McDonald's Corp. of Oak Brook, IL, received the sole honorable mention. The Prizes seek to reward exemplary efforts that uphold and update the ideal of "out of many, one" and inspire others to take on the important work of helping immigrants and their children join the mainstream of U.S. society as well as build stronger ties between immigrants and the native-born. Award winners were honored at the second annual awards ceremony in Washington, DC, on May 18, 2010. Watch highlights of the ceremony here.

This awards ceremony, honoring the 2011 recipients of the E Pluribus Unum Prizes — a national awards program for exceptional immigrant integration initiatives — featured panel discussions with the awardees and federal officials and remarks by White House Director of Intergovernmental Affairs Cecilia Muñoz and Assistant Secretary of Education Brenda Dann-Messier.

This awards ceremony, honoring the 2011 recipients of the E Pluribus Unum Prizes -- a national awards program for exceptional immigrant integration initiatives -- featured panel discussions with the awardees and federal officials and remarks by White House Director of Intergovernmental Affairs Cecilia Muñoz and Assistant Secretary of Education Brenda Dann-Messier.

Marking the release of an MPI report, this webinar examines what the growing intersection between U.S. immigration and child welfare systems means for protection agencies. Speakers also discuss promising child welfare policies and agency approaches to address the needs of children of immigrants and their families amid demographic change and rising immigration enforcement.

Faced with a lack of employment opportunities and recurrent poverty, Albanian youth migrate to Italy alone in the hopes of improving their educational prospects or making money for their families. Yet upon arrival, they face many vulnerabilities. While some protections for unaccompanied minors exist in the Italy, the system is greatly fragmented and challenges, including how to return them to Albania, remain persistent.

The Trump administration’s decision to terminate the Deferred Action for Childhood Arrivals (DACA) deportation-relief program launched in 2012 has sparked new urgency to find a longer-term fix for "DREAMers," the unauthorized immigrants brought to the United States as children. This Policy Beat article examines movement in the courts and in Congress on the DREAM Act and similar proposals, exploring likely paths forward.

In a year when immigration has been prominent in the headlines, the 14th annual Immigration Law and Policy Conference provides timely policy and legal analysis, with expert discussion covering the shifting immigration policy priorities under a new administration, including changes in immigration enforcement, border enforcement, refugee resettlement, the treatment of unaccompanied minors and their families, Temporary Protected Status, and DACA, along with the responses from the judicial and congressional branches, various stakeholders, and state and local governments. 

The Trump administration has been steadily building a case to penalize "sanctuary" cities—those jurisdictions that in some way limit their cooperation with federal immigration authorities—after threatening to cut federal funding. Despite court rulings that seemed to strike a blow against these efforts, the Justice Department is moving forward with its strategy, relying on a broad interpretation of a federal statute, as this article explores.

The Trump administration has significantly cranked up the immigration enforcement machinery in the U.S. interior. Yet even as arrests and deportations are up in the early Trump months, they remain less than half their peaks. This report demonstrates how pushback from California and other "sanctuary" locations makes it quite unlikely that ICE will be able to match record enforcement levels.

Discussion at this event focused on findings from MPI's report examining the interior immigration enforcement system in the United States, including ICE data on deportations and arrests, and the responses of state and local governments, civil society, and consulates.  

Experts on this Spanish language webinar examine the operation of today’s interior immigration enforcement system and how state and local governments, civil society, and consulates are responding.    

At a time of intense and fast-moving action on immigration, 2018's Immigration Law and Policy Conference offered an excellent opportunity to go beyond the headlines with thoughtful analysis from leading experts.

At the 15th annual Immigration Law and Policy Conference, held in October 2018, leading experts and government officials discussed the role that immigration is playing in the mid-term elections; how the courts are handling key immigration questions; and recent changes in the U.S. asylum system, border and interior enforcement, and regulations surrounding legal immigration.   

Remain in Mexico—the Trump administration policy aimed at deterring the rising numbers of migrants from Central America by requiring them to stay in Mexico through most of their U.S. asylum adjudication process—bears striking similarities to U.S. policy in the 1980s and 1990s that sought to discourage Haitians from making the sea journey to the United States. This article explores the parallels and differences between Remain in Mexico and the earlier narrowing of asylum for Haitians.

Approximately 11,500 unaccompanied children were apprehended at the U.S.-Mexico border in May, putting this year on track to exceed 2014's surge. As the U.S. government struggles to care for these child migrants, with public outrage mounting over reports of unsafe, filthy conditions in initial Border Patrol custody, the failure of the executive branch and Congress to plan for increased shelter and care demands are increasingly apparent, as this article explores.

As federal and state governments ramp up efforts to implement the Workforce Innovation and Opportunity Act, these fact sheets compare key characteristics of the foreign born and the U.S. born that are relevant to understanding needs for adult education and workforce training services. The fact sheets cover the United States, the 20 states and 25 counties with the largest immigrant populations, and New York City.

This fact sheet uses U.S. and Mexican apprehensions data to trace the evolving trends in unaccompanied child and family migration from Central America through Mexico and to the United States, and discusses the push factors and pull factors responsible for the increase in flows seen in recent years, as well as the growing role of smuggling organizations.

Growing up with unauthorized immigrant parents puts children—nearly 80 percent of whom were born in the United States—at a disadvantage, with lower preschool enrollment, reduced socioeconomic progress, and higher rates of linguistic isolation and poverty. This fact sheet examines the number, characteristics, and socioeconomic status of children, both U.S.-citizen and noncitizen, who have unauthorized immigrant parents.

The immigrant population in the Kansas City region has grown rapidly over the past 25 years, contributing to overall population growth in the area. This fact sheet describes immigrants in the metro area, examining their origins, industries of employment, income and poverty levels, English proficiency, educational attainment, and more.

These fact sheets provide a sketch of key characteristics of the foreign-born and English Learner (EL) populations in select states. The fact sheets look at the demographics of these states, discuss EL student outcomes as measured by standardized tests, and conclude with an overview of state accountability mechanisms that affect ELs under relevant provisions of the Every Student Succeeds Act and predecessor No Child Left Behind Act.