The Zn2+ transporter ZIP7 enhances endoplasmic-reticulum-associated protein degradation and prevents neurodegeneration in Drosophila.

Proteotoxic stress drives numerous degenerative diseases. Cells initially adapt to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated protein degradation (ERAD). However, persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. The ER-localized Zn2+ transporter ZIP7 is conserved from plants to humans and required for intestinal self-renewal, Notch signaling, cell motility, and survival. However, a unifying mechanism underlying these diverse phenotypes was unknown. In studying Drosophila border cell migration, we discovered that ZIP7-mediated Zn2+ transport enhances the obligatory deubiquitination of proteins by the Rpn11 Zn2+ metalloproteinase in the proteasome lid. In human cells, ZIP7 and Zn2+ are limiting for deubiquitination. In a Drosophila model of neurodegeneration caused by misfolded rhodopsin (Rh1), ZIP7 overexpression degrades misfolded Rh1 and rescues photoreceptor viability and fly vision. Thus, ZIP7-mediated Zn2+ transport is a previously unknown, rate-limiting step for ERAD in vivo with therapeutic potential in protein misfolding diseases.

Apoptotic signaling: Beyond cell death.

Apoptosis is the best described form of regulated cell death, and was, until relatively recently, considered irreversible once particular biochemical points-of-no-return were activated. In this manuscript, we examine the mechanisms cells use to escape from a self-amplifying death signaling module. We discuss the role of feedback, dynamics, propagation, and noise in apoptotic signaling. We conclude with a revised model for the role of apoptosis in animal development, homeostasis, and disease.

Hyperactive Rac stimulates cannibalism of living target cells and enhances CAR-M-mediated cancer cell killing.

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy.

The molecular mechanisms of diapause and diapause-like reversible arrest.

Diapause is a protective mechanism that many organisms deploy to overcome environmental adversities. Diapause extends lifespan and fertility to enhance the reproductive success and survival of the species. Although diapause states have been known and employed for commercial purposes, for example in the silk industry, detailed molecular and cell biological studies are an exciting frontier. Understanding diapause-like protective mechanisms will shed light on pathways that steer organisms through adverse conditions. One hope is that an understanding of the mechanisms that support diapause might be leveraged to extend the lifespan and/or health span of humans as well as species threatened by climate change. In addition, recent findings suggest that cancer cells that persist after treatment mimic diapause-like states, implying that these programs may facilitate cancer cell survival from chemotherapy and cause relapse. Here, we review the molecular mechanisms underlying diapause programs in a variety of organisms, and we discuss pathways supporting diapause-like states in tumor persister cells.

Activated Src kinase promotes cell cannibalism in Drosophila.

Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Here, using the Drosophila melanogaster border cells as a model, we report that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, we found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. We elucidate a molecular mechanism that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. We propose that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior.

Nuclear lamin facilitates collective border cell invasion into confined spaces in vivo.

Cells migrate collectively through confined environments during development and cancer metastasis. The nucleus, a stiff organelle, impedes single cells from squeezing into narrow channels within artificial environments. However, how nuclei affect collective migration into compact tissues is unknown. Here, we use border cells in the fly ovary to study nuclear dynamics in collective, confined in vivo migration. Border cells delaminate from the follicular epithelium and squeeze into tiny spaces between cells called nurse cells. The lead cell nucleus transiently deforms within the lead cell protrusion, which then widens. The nuclei of follower cells deform less. Depletion of the Drosophila B-type lamin, Lam, compromises nuclear integrity, hinders expansion of leading protrusions, and impedes border cell movement. In wildtype, cortical myosin II accumulates behind the nucleus and pushes it into the protrusion, whereas in Lam-depleted cells, myosin accumulates but does not move the nucleus. These data suggest that the nucleus stabilizes lead cell protrusions, helping to wedge open spaces between nurse cells.

Septins regulate border cell surface geometry, shape, and motility downstream of Rho in Drosophila.

Septins self-assemble into polymers that bind and deform membranes in vitro and regulate diverse cell behaviors in vivo. How their in vitro properties relate to their in vivo functions is under active investigation. Here, we uncover requirements for septins in detachment and motility of border cell clusters in the Drosophila ovary. Septins and myosin colocalize dynamically at the cluster periphery and share phenotypes but, surprisingly, do not impact each other. Instead, Rho independently regulates myosin activity and septin localization. Active Rho recruits septins to membranes, whereas inactive Rho sequesters septins in the cytoplasm. Mathematical analyses identify how manipulating septin expression levels alters cluster surface texture and shape. This study shows that the level of septin expression differentially regulates surface properties at different scales. This work suggests that downstream of Rho, septins tune surface deformability while myosin controls contractility, the combination of which governs cluster shape and movement.

Rescue of proteotoxic stress and neurodegeneration by the Zn2+ transporter ZIP7.

Proteotoxic stress drives numerous degenerative diseases. In response to misfolded proteins, cells adapt by activating the unfolded protein response (UPR), including endoplasmic reticulum-associated protein degradation (ERAD). However persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. From plants to humans, loss of the Zn2+ transporter ZIP7 causes ER stress, however the mechanism is unknown. Here we show that ZIP7 enhances ERAD and that cytosolic Zn2+ is limiting for deubiquitination of client proteins by the Rpn11 Zn2+ metalloproteinase as they enter the proteasome in Drosophila and human cells. ZIP7 overexpression rescues defective vision caused by misfolded rhodopsin in Drosophila. Thus ZIP7 overexpression may prevent diseases caused by proteotoxic stress, and existing ZIP inhibitors may be effective against proteasome-dependent cancers.

Who's really in charge: Diverse follower cell behaviors in collective cell migration.

Collective cell migrations drive morphogenesis, wound healing, and cancer dissemination. Cells located at the front are considered leaders while those behind them are defined topologically as followers. Leader cell behaviors, including chemotaxis and their coupling to followers, have been well-studied and reviewed. However, the contributions of follower cells to collective cell migration represent an emerging area of interest. In this perspective, we highlight recent research into the broadening array of follower cell behaviors found in moving collectives. We describe examples of follower cells that possess cryptic leadership potential and followers that lack that potential but contribute in diverse and sometimes surprising ways to collective movement, even steering from behind. We highlight collectives in which all cells both lead and follow, and a few passive passengers. The molecular mechanisms controlling follower cell function and behavior are just emerging and represent an exciting frontier in collective cell migration research.

Cell survival following direct executioner-caspase activation.

Executioner-caspase activation has been considered a point-of-no-return in apoptosis. However, numerous studies report survival from caspase activation after treatment with drugs or radiation. An open question is whether cells can recover from direct caspase activation without pro-survival stress responses induced by drugs. To address this question, we engineered a HeLa cell line to express caspase-3 inducibly and combined it with a quantitative caspase activity reporter. While high caspase activity levels killed all cells and very low levels allowed all cells to live, doses of caspase activity sufficient to kill 15 to 30% of cells nevertheless allowed 70 to 85% to survive. At these doses, neither the rate, nor the peak level, nor the total amount of caspase activity could accurately predict cell death versus survival. Thus, cells can survive direct executioner-caspase activation, and variations in cellular state modify the outcome of potentially lethal caspase activity. Such heterogeneities may underlie incomplete tumor cell killing in response to apoptosis-inducing cancer treatments.

Anastasis confers ovarian cancer cells increased malignancy through elevated p38 MAPK activation.

Activation of executioner caspases was once considered as a point of no return in apoptosis. However, in recent years, accumulating evidence has demonstrated that cells can survive executioner caspase activation in response to apoptotic stimuli through a process called anastasis. In this study, we developed a reporter system, mCasExpress, to track mammalian cells that survive executioner caspase activation. We demonstrate that anastatic ovarian cancer cells acquire enhanced migration following their transient exposure to apoptotic stimulus TRAIL or Paclitaxel. Moreover, anastatic cancer cells secrete more pro-angiogenic factors that enable tumor angiogenesis, growth and metastasis. Mechanistically, we demonstrate that activation of p38 MAPK, which occurs in a caspase-dependent manner in response to apoptotic stress to promote anastasis, persists at a higher level in anastatic cancer cells even after removal of apoptotic stimuli. Importantly, p38 is essential for the elevated migratory and angiogenic capacity in the anastatic cells. Our work unveils anastasis as a potential driver of tumor angiogenesis and metastasis.

A Scribble/Cdep/Rac pathway controls follower-cell crawling and cluster cohesion during collective border-cell migration.

Collective cell movements drive normal development and metastasis. Drosophila border cells move as a cluster of 6-10 cells, where the role of the Rac GTPase in migration was first established. In border cells, as in most migratory cells, Rac stimulates leading-edge protrusion. Upstream Rac regulators in leaders have been identified; however, the regulation and function of Rac in follower border cells is unknown. Here, we show that all border cells require Rac, which promotes follower-cell motility and is important for cluster compactness and movement. We identify a Rac guanine nucleotide exchange factor, Cdep, which also regulates follower-cell movement and cluster cohesion. Scribble, Discs large, and Lethal giant larvae localize Cdep basolaterally and share phenotypes with Cdep. Relocalization of Cdep::GFP partially rescues Scribble knockdown, suggesting that Cdep is a major downstream effector of basolateral proteins. Thus, a Scrib/Cdep/Rac pathway promotes cell crawling and coordinated, collective migration in vivo.

Macrophages, masters of invasion.

Macrophages are exceptionally invasive cells. In a recent article in Science, Akhmanova et al. describe a novel mechanism facilitating macrophage infiltration into a tightly packed tissue in Drosophila embryos. Ectodermal cell rounding and division enhance macrophage entry by detaching the dividing cells from the underlying extracellular matrix.

Independently paced Ca2+ oscillations in progenitor and differentiated cells in an ex vivo epithelial organ.

Cytosolic Ca2+ is a highly dynamic, tightly regulated and broadly conserved cellular signal. Ca2+ dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. Here, we examine Ca2+ dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem-cell daughters adopt strikingly distinct patterns of Ca2+ oscillations after differentiation: enteroendocrine cells exhibit single-cell Ca2+ oscillations, whereas enterocytes exhibit rhythmic, long-range Ca2+ waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), of which the oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates Ca2+ oscillations in all cell types - even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of Ca2+ dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.

Border cell polarity and collective migration require the spliceosome component Cactin.

Border cells are an in vivo model for collective cell migration. Here, we identify the gene cactin as essential for border cell cluster organization, delamination, and migration. In Cactin-depleted cells, the apical proteins aPKC and Crumbs (Crb) become abnormally concentrated, and overall cluster polarity is lost. Apically tethering excess aPKC is sufficient to cause delamination defects, and relocalizing apical aPKC partially rescues delamination. Cactin is conserved from yeast to humans and has been implicated in diverse processes. In border cells, Cactin's evolutionarily conserved spliceosome function is required. Whole transcriptome analysis revealed alterations in isoform expression in Cactin-depleted cells. Mutations in two affected genes, Sec23 and Sec24CD, which traffic Crb to the apical cell surface, partially rescue border cell cluster organization and migration. Overexpression of Rab5 or Rab11, which promote Crb and aPKC recycling, similarly rescues. Thus, a general splicing factor is specifically required for coordination of cluster polarity and migration, and migrating border cells are particularly sensitive to splicing and cell polarity disruptions.

Enhanced germline stem cell longevity in Drosophila diapause.

In many species including humans, aging reduces female fertility. Intriguingly, some animals preserve fertility longer under specific environmental conditions. For example, at low temperature and short day-length, Drosophila melanogaster enters a state called adult reproductive diapause. As in other stressful conditions, ovarian development arrests at the yolk uptake checkpoint; however, mechanisms underlying fertility preservation and post-diapause recovery are largely unknown. Here, we report that diapause causes more complete arrest than other stresses yet preserves greater recovery potential. During dormancy, germline stem cells (GSCs) incur DNA damage, activate p53 and Chk2, and divide less. Despite reduced niche signaling, germline precursor cells do not differentiate. GSCs adopt an atypical, suspended state connected to their daughters. Post-diapause recovery of niche signaling and resumption of division contribute to restoring GSCs. Mimicking one feature of quiescence, reduced juvenile hormone production, enhanced GSC longevity in non-diapausing flies. Thus, diapause mechanisms provide approaches to GSC longevity enhancement.

Unconventional translation initiation factor EIF2A is required for Drosophila spermatogenesis.

BACKGROUND: EIF2A is an unconventional translation factor required for initiation of protein synthesis from non-AUG codons from a variety of transcripts, including oncogenes and stress related transcripts in mammalian cells. Its function in multicellular organisms has not been reported. RESULTS: Here, we identify and characterize mutant alleles of the CG7414 gene, which encodes the Drosophila EIF2A ortholog. We identified that CG7414 undergoes sex-specific splicing that regulates its male-specific expression. We characterized a Mi{Mic} transposon insertion that disrupts the coding regions of all predicted isoforms and is a likely null allele, and a PBac transposon insertion into an intron, which is a hypomorph. The Mi{Mic} allele is homozygous lethal, while the viable progeny from the hypomorphic PiggyBac allele are male sterile and female fertile. In dEIF2A mutant flies, sperm failed to individualize due to defects in F-actin cones and failure to form and maintain cystic bulges, ultimately leading to sterility. CONCLUSIONS: These results demonstrate that EIF2A is essential in a multicellular organism, both for normal development and spermatogenesis, and provide an entrée into the elucidation of the role of EIF2A and unconventional translation in vivo.

EMT, One of Many Morphological Transitions in Cellular Phase Space.

The epithelial to mesenchymal transition (EMT) is an enticingly simple mechanism that converts stationary epithelial cells into migratory mesenchymal cells. EMT is meant to provide a unified explanation for phenomena as complex as gastrulation and metastasis. However, cell movements turn out to be diverse, and many are collective. Cells commonly migrate in clusters, strands, sheets, elongating tubes, or in fluid-like masses. Moreover, plenty of cells move without activating the EMT program. Here I propose that EMT can be understood as one of many types of transitions in a broader landscape-or phase space-of cell morphologies and behaviors. Throughout biology, and at multiple scales, complexity arises from the combinatorial deployment of simple, modular components. I propose that diversity of cell shapes and behaviors similarly arises from combinatorial use of modular biomechanical properties.

Akt1 and dCIZ1 promote cell survival from apoptotic caspase activation during regeneration and oncogenic overgrowth.

Apoptosis is an ancient and evolutionarily conserved cell suicide program. During apoptosis, executioner caspase enzyme activation has been considered a point of no return. However, emerging evidence suggests that some cells can survive caspase activation following exposure to apoptosis-inducing stresses, raising questions as to the physiological significance and underlying molecular mechanisms of this unexpected phenomenon. Here, we show that, following severe tissue injury, Drosophila wing disc cells that survive executioner caspase activation contribute to tissue regeneration. Through RNAi screening, we identify akt1 and a previously uncharacterized Drosophila gene CG8108, which is homologous to the human gene CIZ1, as essential for survival from the executioner caspase activation. We also show that cells expressing activated oncogenes experience apoptotic caspase activation, and that Akt1 and dCIZ1 are required for their survival and overgrowth. Thus, survival following executioner caspase activation is a normal tissue repair mechanism usurped to promote oncogene-driven overgrowth.