(searched for: doi:10.1042/bss0740095)
Nature Communications, Volume 13, pp 1-17; https://doi.org/10.1038/s41467-021-27748-w
Cell polarity is a fundamental feature underlying cell morphogenesis and organismal development. In the Arabidopsis stomatal lineage, the polarity protein BASL controls stomatal asymmetric cell division. However, the cellular machinery by which this intrinsic polarity site is established remains unknown. Here, we identify the PRAF/RLD proteins as BASL physical partners and mutating four PRAF members leads to defects in BASL polarization. Members of PRAF proteins are polarized in stomatal lineage cells in a BASL-dependent manner. Developmental defects of the praf mutants phenocopy those of the gnom mutants. GNOM is an activator of the conserved Arf GTPases and plays important roles in membrane trafficking. We further find PRAF physically interacts with GNOM in vitro and in vivo. Thus, we propose that the positive feedback of BASL and PRAF at the plasma membrane and the connected function of PRAF and GNOM in endosomal trafficking establish intrinsic cell polarity in the Arabidopsis stomatal lineage.
Published: 11 August 2020
Frontiers in Cell and Developmental Biology, Volume 8; https://doi.org/10.3389/fcell.2020.00779
Intracellular trafficking is essential for cell structure and function. In order to perform key tasks such as phagocytosis, secretion or migration, cells must coordinate their intracellular trafficking and cytoskeleton dynamics. This relies on certain classes of proteins endowed with specialized and conserved domains that bridge membranes with effector proteins. Of particular interest are proteins capable of interacting with membrane subdomains enriched in specific phosphatidylinositol lipids, tightly regulated by various kinases and phosphatases. Here, we focus on the poorly studied RUFY family of adaptor proteins, characterized by a RUN domain, which interacts with small GTP-binding proteins, and a FYVE domain, involved in the recognition of phosphatidylinositol 3-phosphate. We report recent findings on this protein family that regulates endosomal trafficking, cell migration and upon dysfunction, can lead to severe pathology at the organismal level.
Infection and Immunity, Volume 87; https://doi.org/10.1128/iai.00153-19
Legionella pneumophila and other Legionella species replicate intracellularly using the Icm/Dot type IV secretion system. In L. pneumophila this system translocates >300 effectors into host cells and in the Legionella genus thousands of effectors were identified, the function of most of which is unknown.
Oncogene, Volume 37, pp 5367-5386; https://doi.org/10.1038/s41388-018-0330-0
Serine–threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt–Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight–mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine–threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2–Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2–Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2–Akt complexes in the lysosomes via modulation of autophagy.
Frontiers in Microbiology, Volume 6; https://doi.org/10.3389/fmicb.2015.00001
Fungal secondary metabolism has become an important research topic with great biomedical and biotechnological value. In the postgenomic era, understanding the diversity and the molecular control of secondary metabolites are two challenging tasks addressed by the research community. Discovery of the LaeA methyltransferase 10 years ago opened up a new horizon on the control of secondary metabolite research when it was found that expression of many secondary metabolite gene clusters is controlled by LaeA. While the molecular function of LaeA remains an enigma, discovery of the velvet family proteins as interaction partners further extended the role of the LaeA beyond secondary metabolism. The heterotrimeric VelB-VeA-LaeA complex plays important roles in development, sporulation, secondary metabolism and pathogenicity. Recently, three other methyltransferases have been found to associate with the velvet complex, the LaeA-like methyltransferase F (LlmF) and the methyltransferase heterodimers VipC-VapB. Interaction of VeA with at least four methyltransferase proteins indicates a molecular hub function for VeA that questions: Is there a VeA supercomplex or is VeA part of a highly dynamic cellular control network with many different partners?
Orphanet Journal of Rare Diseases, Volume 9, pp 1-11; https://doi.org/10.1186/s13023-014-0141-5
The publisher has not yet granted permission to display this abstract.
Journal of Neuroscience, Volume 34, pp 373-391; https://doi.org/10.1523/jneurosci.0876-13.2014
The elongation rate of axons is tightly regulated during development. Recycling of the plasma membrane is known to regulate axon extension; however, the specific molecules involved in recycling within the growth cone have not been fully characterized. Here, we investigated whether the small GTPases Rab4 and Rab5 involved in short-loop recycling regulate the extension of Xenopus retinal axons. We report that, in growth cones, Rab5 and Rab4 proteins localize to endosomes, which accumulate markers that are constitutively recycled. Fluorescence recovery after photo-bleaching experiments showed that Rab5 and Rab4 are recruited to endosomes in the growth cone, suggesting that they control recycling locally. Dynamic image analysis revealed that Rab4-positive carriers can bud off from Rab5 endosomes and move to the periphery of the growth cone, suggesting that both Rab5 and Rab4 contribute to recycling within the growth cone. Inhibition of Rab4 function with dominant-negative Rab4 or Rab4 morpholino and constitutive activation of Rab5 decreases the elongation of retinal axons in vitro and in vivo, but, unexpectedly, does not disrupt axon pathfinding. Thus, Rab5- and Rab4-mediated control of endosome trafficking appears to be crucial for axon growth. Collectively, our results suggest that recycling from Rab5-positive endosomes via Rab4 occurs within the growth cone and thereby supports axon elongation.
Experimental parasitology, Volume 133, pp 255-264; https://doi.org/10.1016/j.exppara.2012.11.007
► TbFRP is a GTPase with a novel architecture (FYVE-GTPase). ► Orthologs of TbFRP are restricted to Euglenozoa. ► TbFRP is rapidly cleaved to release the GTPase domain. ► The TbFRP FYVE domain is functional in PIP3-binding. ► Expression levels of TbFRP are controlled in a cell density dependent manner.
International Journal of Molecular Sciences, Volume 14, pp 6487-6498; https://doi.org/10.3390/ijms14036487
Cell survival, homeostasis and cell polarity rely on the control of membrane trafficking pathways. The RUN domain (comprised of the RPIP8, UNC-14, and NESCA proteins) has been suggested to be implicated in small GTPase-mediated membrane trafficking and cell polarity. Accumulating evidence supports the hypothesis that the RUN domain-containing proteins might be responsible for an interaction with a filamentous network linked to actin cytoskeleton and/or microtubules. In addition, several downstream molecules of PI3K are involved in regulation of the membrane trafficking by interacting with vesicle-associated RUN proteins such as RUFY family proteins. In this review, we summarize the background of RUN domain research with an emphasis on the interaction between RUN domain proteins including RUFY proteins (designated as RUN and FYVE domain-containing proteins) and several small GTPases with respect to the regulation of cell polarity and membrane trafficking on filamentous network
Frontiers in Immunology, Volume 3; https://doi.org/10.3389/fimmu.2012.00037
Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.
Journal of cell science, Volume 125, pp 3265-3270; https://doi.org/10.1242/jcs.093419
Disabling mutations in the FGD1 gene cause faciogenital dysplasia (also known as Aarskog-Scott syndrome), a human X-linked developmental disorder that results in disproportionately short stature, facial, skeletal and urogenital anomalies, and in a number of cases, mild mental retardation. FGD1 encodes the guanine nucleotide exchange factor FGD1, which is specific for the Rho GTPase cell division cycle 42 (CDC42). CDC42 controls cytoskeleton-dependent membrane rearrangements, transcriptional activation, secretory membrane trafficking, G1 transition during the cell cycle and tumorigenic transformation. The cellular mechanisms by which FGD1 mutations lead to the hallmark skeletal deformations of faciogenital dysplasia remain unclear, but the pathology of the disease, as well as some recent discoveries, clearly show that the protein is involved in the regulation of bone development. Two recent studies unveiled new potential functions of FGD1, in particular, its involvement in the regulation of the formation and function of invadopodia and podosomes, which are cellular structures devoted to degradation of the extracellular matrix in tumour and endothelial cells. Here, we discuss the hypothesis that FGD1 might be an important regulator of events controlling extracellular matrix remodelling and possibly cell invasion in physiological and pathological settings. Additionally, we focus on how studying the cell biology of FGD1 might help us to connect the dots that link CDC42 signalling with remodelling of the extracellular matrix (ECM) in physiology and complex diseases, while, at the same time, furthering our understanding of the pathogenesis of faciogenital dysplasia.
PLOS ONE, Volume 6; https://doi.org/10.1371/journal.pone.0029584
ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27ΔHR3 failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27ΔHR3 was co-expressed with wild-type ZFYVE27 (ZFYVE27WT), it exerted a dominant negative effect on ZFYVE27WT as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions.
Molecular Omics, Volume 7, pp 2261-2277; https://doi.org/10.1039/c1mb05061c
Recent studies point to a diverse assemblage of prokaryotic cognates of the eukaryotic ubiquitin (Ub) system. These systems span an entire spectrum, ranging from those catalyzing cofactor and amino acid biosynthesis, with only adenylating E1-like enzymes and ubiquitin-like proteins (Ubls), to those that are closer to eukaryotic systems by virtue of possessing E2 enzymes. Until recently E3 enzymes were unknown in such prokaryotic systems. Using contextual information from comparative genomics, we uncover a diverse group of RING finger E3s in prokaryotes that are likely to function with E1s, E2s, JAB domain peptidases and Ubls. These E1s, E2s and RING fingers suggest that features hitherto believed to be unique to eukaryotic versions of these proteins emerged progressively in such prokaryotic systems. These include the specific configuration of residues associated with oxyanion-hole formation in E2s and the C-terminal UFD in the E1 enzyme, which presents the E2 to its active site. Our study suggests for the first time that YukD-like Ubls might be conjugated by some of these systems in a manner similar to eukaryotic Ubls. We also show that prokaryotic RING fingers possess considerable functional diversity and that not all of them are involved in Ub-related functions. In eukaryotes, other than RING fingers, a number of distinct binuclear (chelating two Zn atoms) and mononuclear (chelating one zinc atom) treble clef domains are involved in Ub-related functions. Through detailed structural analysis we delineated the higher order relationships and interaction modes of binuclear treble clef domains. This indicated that the FYVE domain acquired the binuclear state independently of the other binuclear forms and that different treble clef domains have convergently acquired Ub-related functions independently of the RING finger. Among these, we uncover evidence for notable prokaryotic radiations of the ZF-UBP, B-box, AN1 and LIM clades of treble clef domains and present contextual evidence to support their role in functions unrelated to the Ub-system in prokaryotes. In particular, we show that bacterial ZF-UBP domains are part of a novel cyclic nucleotide-dependent redox signaling system, whereas prokaryotic B-box, AN1 and LIM domains have related functions as partners of diverse membrane-associated peptidases in processing proteins. This information, in conjunction with structural analysis, suggests that these treble clef domains might have been independently recruited to the eukaryotic Ub-system due to an ancient conserved mode of interaction with peptides.
Molecular microbiology, Volume 79, pp 50-62; https://doi.org/10.1111/j.1365-2958.2010.07429.x
The publisher has not yet granted permission to display this abstract.
Nature, Volume 464, pp 243-249; https://doi.org/10.1038/nature08779
Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-β and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.
PLOS ONE, Volume 5; https://doi.org/10.1371/journal.pone.0008769
Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively. Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant. Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.
Published: 1 January 2010
Journal of lipid research, Volume 50, pp 1245-1254; https://doi.org/10.1194/jlr.d800043-jlr200
A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.
Published: 1 January 2009
Polarity is a prerequisite for proper development and function of epithelia in metazoa. The major feature of polarized epithelial cells is the presence of specialized domains with asymmetric distribution of macromolecular contents including proteins and lipids. The apical domain is involved in exchange with the organ lumen, and the basolateral membrane maintains contact with neighboring cells and the underlying extracellular matrix. The two domains are separated by tight junctions, which act as a diffusion barrier to prevent free mixing of domain-specific proteins and lipids. Extensive studies have shed light on the numerous protein families involved in cell polarization. However, many questions still remain regarding the molecular mechanisms of polarity regulation and in particular very little is known about the role of lipids in building polarity. In this chapter, essential determinants of epithelial polarity will be reviewed with a particular focus on metabolism and function of phosphoinositides.