Polarized protein transport and lumen formation during epithelial tissue morphogenesis.

One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo.

Mutations in cdon and boc affect trunk neural crest cell migration and slow-twitch muscle development in zebrafish.

The transmembrane proteins cdon and boc are implicated in regulating hedgehog signaling during vertebrate development. Recent work showing roles for these genes in axon guidance and neural crest cell migration suggest that cdon and boc may play additional functions in regulating directed cell movements. We use newly generated and existing mutants to investigate a role for cdon and boc in zebrafish neural crest cell migration. We find that single mutant embryos exhibit normal neural crest phenotypes, but that neural crest migration is strikingly disrupted in double cdon;boc mutant embryos. We further show that this migration phenotype is associated with defects in the differentiation of slow-twitch muscle cells, and the loss of a Col1a1a-containing extracellular matrix, suggesting that neural crest defects may be a secondary consequence to defects in mesoderm development. Combined, our data add to a growing literature showing that cdon and boc act synergistically to promote hedgehog signaling during vertebrate development, and suggest that the zebrafish can be used to study the function of hedgehog receptor paralogs.

TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells.

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as axonemal microtubule growth and stabilization in polarized epithelia, are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the α/ß-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

Roles of the actin cytoskeleton in ciliogenesis.

Primary cilia play a key role in the ability of cells to respond to extracellular stimuli, such as signaling molecules and environmental cues. These sensory organelles are crucial to the development of many organ systems, and defects in primary ciliogenesis lead to multisystemic genetic disorders, known as ciliopathies. Here, we review recent advances in the understanding of several key aspects of the regulation of ciliogenesis. Primary ciliogenesis is thought to take different pathways depending on cell type, and some recent studies shed new light on the cell-type-specific mechanisms regulating ciliogenesis at the apical surface in polarized epithelial cells, which are particularly relevant for many ciliopathies. Furthermore, recent findings have demonstrated the importance of actin cytoskeleton dynamics in positively and negatively regulating multiple stages of ciliogenesis, including the vesicular trafficking of ciliary components and the positioning and docking of the basal body. Finally, studies on the formation of motile cilia in multiciliated epithelial cells have revealed requirements for actin remodeling in this process too, as well as showing evidence of an additional alternative ciliogenesis pathway.

CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

During mitotic cell division, the actomyosin cytoskeleton undergoes several dynamic changes that play key roles in progression through mitosis. Although the regulators of cytokinetic ring formation and contraction are well established, proteins that regulate cortical stability during anaphase and telophase have been understudied. Here, we describe a role for CLIC4 in regulating actin and actin regulators at the cortex and cytokinetic cleavage furrow during cytokinesis. We first describe CLIC4 as a new component of the cytokinetic cleavage furrow that is required for successful completion of mitotic cell division. We also demonstrate that CLIC4 regulates the remodeling of the sub-plasma-membrane actomyosin network within the furrow by recruiting MST4 kinase (also known as STK26) and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in regulating cortex stiffness and blebbing during the late stages of cytokinetic furrowing.

Insane in the apical membrane: Trafficking events mediating apicobasal epithelial polarity during tube morphogenesis.

The creation of cellular tubes is one of the most vital developmental processes, resulting in the formation of most organ types. Cells have co-opted a number of different mechanisms for tube morphogenesis that vary among tissues and organisms; however, generation and maintenance of cell polarity is fundamental for successful lumenogenesis. Polarized membrane transport has emerged as a key driver not only for establishing individual epithelial cell polarity, but also for coordination of epithelial polarization during apical lumen formation and tissue morphogenesis. In recent years, much work has been dedicated to identifying membrane trafficking regulators required for lumenogenesis. In this review we will summarize the findings from the past couple of decades in defining the molecular machinery governing lumenogenesis both in 3D tissue culture models and during organ development in vivo.

FYCO1 regulates accumulation of post-mitotic midbodies by mediating LC3-dependent midbody degradation.

The post-mitotic midbody (MB) is a remnant of cytokinesis that can be asymmetrically inherited by one of the daughter cells following cytokinesis. Until recently, the MB was thought to be degraded immediately following cytokinesis. However, recent evidence suggests that the MB is a protein-rich organelle that accumulates in stem cell and cancer cell populations, indicating that it may have post-mitotic functions. Here, we investigate the role of FYCO1, an LC3-binding protein (herein, LC3 refers to MAP1LC3B), and its function in regulating the degradation of post-mitotic MBs. We show that FYCO1 is responsible for formation of LC3-containing membrane around the post-mitotic MB and that FYCO1 knockdown increases MB accumulation. Although MBs accumulate in the stem-cell-like population of squamous cell carcinomas, FYCO1 depletion does not affect the clonogenicity of these cells. Instead, MB accumulation leads to an increase in anchorage-independent growth and invadopodia formation in HeLa cells and squamous carcinoma cells. Collectively, our data suggest that FYCO1 regulates MB degradation, and we present the first evidence that cancer invasiveness is a feature that can be modulated by the accumulation of MBs in cancer stem cells.This article has an associated First Person interview with the first author of the paper.

The role and regulation of Rab40b-Tks5 complex during invadopodia formation and cancer cell invasion.

Invadopodia formation and extracellular matrix degradation are key events during cancer cell invasion, yet little is known about mechanisms mediating these processes. Here, we report that Rab40b plays a key role in mediating invadopodia function during breast cancer cell invasion. We also identify Tks5 (also known as SH3PXD2A), a known Src kinase substrate, as a new Rab40b effector protein and show that Tks5 functions as a tether that mediates Rab40b-dependent targeting of transport vesicles containing MMP2 and MMP9 to the extending invadopodia. Importantly, we also demonstrate that Rab40b and Tks5 levels are regulated by known tumor suppressor microRNA miR-204. This is the first study that identifies a new Rab40b-Tks5- and miR-204-dependent invadopodia transport pathway that regulates MMP2 and MMP9 secretion, and extracellular matrix remodeling during cancer progression.

TRIM17 contributes to autophagy of midbodies while actively sparing other targets from degradation.

TRIM proteins contribute to selective autophagy, a process whereby cells target specific cargo for autophagic degradation. In a previously reported screen, TRIM17 acted as a prominent inhibitor of bulk autophagy, unlike the majority of TRIMs, which had positive roles. Nevertheless, TRIM17 showed biochemical hallmarks of autophagy-inducing TRIMs. To explain this paradox, here, we investigated how TRIM17 inhibits selective autophagic degradation of a subset of targets while promoting degradation of others. We traced the inhibitory function of TRIM17 to its actions on the anti-autophagy protein Mcl-1, which associates with and inactivates Beclin 1. TRIM17 expression stabilized Mcl-1-Beclin-1 complexes. Despite its ability to inhibit certain types of selective autophagy, TRIM17 promoted the removal of midbodies, remnants of the cell division machinery that are known autophagy targets. The selective loss of anti-autophagy Mcl-1 from TRIM17-Beclin-1 complexes at midbodies correlated with the ability of TRIM17 to promote midbody removal. This study further expands the roles of TRIMs in regulating selective autophagy by showing that a single TRIM can, depending upon a target, either positively or negatively regulate autophagy.

Midbody: From the Regulator of Cytokinesis to Postmitotic Signaling Organelle.

Faithful cell division is crucial for successful proliferation, differentiation, and development of cells, tissue homeostasis, and preservation of genomic integrity. Cytokinesis is a terminal stage of cell division, leaving two genetically identical daughter cells connected by an intercellular bridge (ICB) containing the midbody (MB), a large protein-rich organelle, in the middle. Cell division may result in asymmetric or symmetric abscission of the ICB. In the first case, the ICB is severed on the one side of the MB, and the MB is inherited by the opposite daughter cell. In the second case, the MB is cut from both sides, expelled into the extracellular space, and later it can be engulfed by surrounding cells. Cells with lower autophagic activity, such as stem cells and cancer stem cells, are inclined to accumulate MBs. Inherited MBs affect cell polarity, modulate intra- and intercellular communication, enhance pluripotency of stem cells, and increase tumorigenic potential of cancer cells. In this review, we briefly summarize the latest knowledge on MB formation, inheritance, degradation, and function, and in addition, present and discuss our recent findings on the electrical and chemical communication of cells connected through the MB-containing ICB.

FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis.

Apical lumen formation is a key step during epithelial morphogenesis. The establishment of the apical lumen is a complex process that involves coordinated changes in plasma membrane composition, endocytic transport, and cytoskeleton organization. These changes are accomplished, at least in part, by the targeting and fusion of Rab11/FIP5-containing apical endosomes with the apical membrane initiation site (AMIS). Although AMIS formation and polarized transport of Rab11/FIP5-containing endosomes are crucial for the formation of a single apical lumen, the spatiotemporal regulation of this process remains poorly understood. Here, we demonstrate that the formation of the midbody during cytokinesis is a symmetry-breaking event that establishes the location of the AMIS. The interaction of FIP5 with SNX18, which is required for the formation of apical endocytic carriers, is inhibited by GSK-3 phosphorylation at FIP5-T276. Importantly, we show that FIP5-T276 phosphorylation occurs specifically during metaphase and anaphase, to ensure the fidelity and timing of FIP5-endosome targeting to the AMIS during apical lumen formation.

Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells.

Invadopodia-dependent degradation of the basement membrane plays a major role during metastasis of breast cancer cells. Basement membrane degradation is mediated by targeted secretion of various matrix metalloproteinases (MMPs). Specifically, MMP2 and MMP9 (MMP2/9) possess the ability to hydrolyze components of the basement membrane and regulate various aspects of tumor growth and metastasis. However, the membrane transport machinery that mediates targeting of MMP2/9 to the invadopodia during cancer cell invasion remains to be defined. Because Rab GTPases are key regulators of membrane transport, we screened a human Rab siRNA library and identified Rab40b GTPase as a protein required for secretion of MMP2/9. We also have shown that Rab40b functions during at least two distinct steps of MMP2/9 transport. Here, we demonstrate that Rab40b is required for MMP2/9 sorting into VAMP4-containing secretory vesicles. We also show that Rab40b regulates transport of MMP2/9 secretory vesicles during invadopodia formation and is required for invadopodia-dependent extracellular matrix degradation. Finally, we demonstrate that Rab40b is also required for breast cancer cell invasion in vitro. On the basis of these findings, we propose that Rab40b mediates trafficking of MMP2/9 during invadopodia formation and metastasis of breast cancer cells.

Collagen architecture in pregnancy-induced protection from breast cancer.

The reduction in breast cancer risk attributed to early-age pregnancy is mediated in part by changes in the mammary epithelium. Here, we address the role of the mammary stroma in this protection. Utilizing tumor cells capable of transitioning from indolent to proliferative or invasive states, we demonstrate that mammary extracellular matrix (ECM) from parous rats (parous matrix) decreases tumor growth and impedes cellular phenotypes associated with tumor cell invasion compared with that observed using nulliparous matrix. Proteomic analysis identifies an increased abundance of collagen I in parous matrix, an observation extended to breast tissue of parous women. Given the pro-tumorigenic attributes of fibrillar collagen, these results were unexpected. Second-harmonic generation imaging and atomic force microscopy revealed that the abundant collagen observed in the mammary glands of parous rats is less linearized and associated with a decrease in stromal stiffness, implicating collagen organization and stiffness in parity-induced protection. Using 3D cell culture models, we demonstrate that linearized (fibrillar) collagen I induces cellular phenotypes consistent with an invasive behavior in mammary tumor cells and alters the subcellular distribution of ß1 integrin. Conversely, high-density non-fibrillar collagen I induces tumor-suppressive attributes, including increases in junctional E-cadherin in tumor cells, upregulation of genes encoding components of cell-cell junctions, and downregulation of mesenchymal-specific and metalloproteinase-encoding genes. These data show that collagen organization, rather than density alone, is a key contributor to the invasive phenotype. Furthermore, our data show that parity alters the composition and organization of mammary ECM, particularly fibrillar collagen, in a manner consistent with tumor suppression.

Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling.

There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS.

Spatiotemporal inhibition of innate immunity signaling by the Tbc1d23 RAB-GAP.

We previously identified Tbc1d23 as a candidate novel regulator of innate immunity using comparative genomics RNA interference screens in Caenorhabditis elegans and mouse macrophages. Using Tbc1d23 knockout mice and macrophages engineered to overexpress Tbc1d23, we now show that Tbc1d23 is a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin-signaling pathways. Tbc1d23 likely acts downstream of the TLR-signaling adaptors MyD88 and Trif and upstream of the transcription factor XBP1. Importantly, like XBP1, Tbc1d23 affects the maintenance, but not the initiation, of inflammatory cytokine production induced by LPS. Tbc1d23 acts as a RAB-GAP to regulate innate immunity signaling. Thus, Tbc1d23 exerts its inhibitory effect on innate immunity signaling in a spatiotemporal fashion. The identification of a novel spatiotemporal regulator of innate immunity signaling validates the comparative genomics approach for innate immunity gene discovery.

Phosphorylation-dependent allosteric regulation of Cx43 gap junction inhibitor potency.

Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication through gap junctions (GJIC). Connexin (Cx)-based GJ channels are formed of two apposing hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. The main modulators of GJ conductance are transjunctional voltage, intracellular pH, Ca2+, Mg2+, and phosphorylation. Chemical modulators of GJIC are being used in cases of various intercellular communication-dependent diseases. In this study, we used molecular docking, dual whole-cell patch-clamp, and Western blotting to investigate the impact of connexin phosphorylation on GJ chemical gating by α-pinene and other GJ inhibitors (octanol, carbenoxolone, mefloquine, intracellular pH, glycyrrhetinic acid, and sevoflurane) in HeLa cells expressing exogenous Cx43 (full length and truncated at amino acid 258) and other connexins typical of heart and/or nervous system (Cx36, Cx40, Cx45, and Cx47), and in cells expressing endogenous Cx43 (Novikoff and U-87). We found that Ca2+-regulated kinases, such as Ca2+/calmodulin-dependent kinase II, atypical protein kinase C, cyclin-dependent kinase, and Pyk2 kinase may allosterically modulate the potency of α-pinene through phosphorylation of Cx43 C-terminus. The identified new phenomenon was Cx isoform-, inhibitor-, and cell type-dependent. Overall, these results suggest that compounds, the potency of which depends on receptor phosphorylation, might be of particular interest in developing targeted therapies for diseases accompanied by high kinase activity, such as cardiac arrhythmias, epilepsy, stroke, essential tremor, inflammation, and cancer.

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4.

Dysfunctions of primary cilia and cilia-derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans-Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase-activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post-TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1-mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4-based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

Endocytic membrane fusion and buckling-induced microtubule severing mediate cell abscission.

Cytokinesis and abscission are complicated events that involve changes in membrane transport and cytoskeleton organization. We have used the combination of time-lapse microscopy and correlative high-resolution 3D tomography to analyze the regulation and spatio-temporal remodeling of endosomes and microtubules during abscission. We show that abscission is driven by the formation of a secondary ingression within the intracellular bridge connecting two daughter cells. The initiation and expansion of this secondary ingression requires recycling endosome fusion with the furrow plasma membrane and nested central spindle microtubule severing. These changes in endosome fusion and microtubule reorganization result in increased intracellular bridge plasma membrane dynamics and abscission. Finally, we show that central spindle microtubule reorganization is driven by localized microtubule buckling and breaking, rather than by spastin-dependent severing. Our results provide a new mechanism for mediation and regulation of the abscission step of cytokinesis.

HOPS-dependent lysosomal fusion controls Rab19 availability for ciliogenesis in polarized epithelial cells.

Primary cilia are sensory cellular organelles crucial for organ development and homeostasis. Ciliogenesis in polarized epithelial cells requires Rab19-mediated clearing of apical cortical actin to allow the cilium to grow from the apically-docked basal body into the extracellular space. Loss of the lysosomal membrane-tethering HOPS complex disrupts this actin-clearing and ciliogenesis, but it remains unclear how ciliary function of HOPS relates to its canonical function in regulating late endosome-lysosome fusion. Here, we show that disruption of HOPS-dependent lysosomal fusion indirectly impairs actin-clearing and ciliogenesis by disrupting the targeting of Rab19 to the basal body. We also find that Rab19 functions in endolysosomal cargo trafficking apart from its previously-identified role in ciliogenesis. In summary, we show that inhibition of lysosomal fusion abnormally accumulates Rab19 on late endosomes, thus depleting Rab19 from the basal body and thereby disrupting Rab19-mediated actin-clearing and ciliogenesis. Summary statement: Loss of HOPS-mediated lysosomal fusion indirectly blocks apical actin clearing and ciliogenesis in polarized epithelia by trapping Rab19 on late endosomes and depleting Rab19 from the basal body.