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Fluorescence bioimaging of signal transduction dynamics


The Ras-PI3K signaling pathway mediates clathrin-independent endocytosis

The Ras proteins are monomeric GTPases that regulate a variety of signal transduction cascades. More than ten different effectors have been identified, and this variety is responsible for the many facets of Ras functions. The precise mechanism by which Ras exploits different effectors remains poorly understood, however. Spatial regulation of Ras may constitute one of the possible mechanisms through which Ras elicits heterogeneous signals by binding to different effectors in distinct cellular locations.
To test this hypothesis, we analyzed the binding of Ras to its effector molecules in living cells by using a BiFC technique, and demonstrated that the complexes of Ras and the lipid kinase phosphoinositide 3-kinase (PI3K) were preferentially formed not only at the plasma membrane but also in the endosomal compartments (Figure 1). In contrast, other effector molecules, including Raf serine-threonine kinase and Ral guanine nucleotide dissociation stimulator (RalGDS), bound to Ras at the plasma membrane. This study highlights the importance of Ras-PI3K signaling from endosomes, which has been thought to work mainly at the plasma membrane. Indeed, we also reported that Ras-PI3K signaling from endosomes mediates clathrin-independent endocytosis.
Given that these discoveries were brought about by live cell imaging, we deem that it might be a fine exemple of “seeing is believing”. We are now exploring the mechanisms underlying the regulation of endocytosis by Ras-PI3K signaling.




【Related papers】

  • Visualization of Ras-PI3K interaction in the endosome using BiFC. K. Tsutsumi, Y. Fujioka, M. Tsuda,H. Kawaguchi, Y. Ohba. Cell Signal. 21(11): 1672-1679 (2009)

  • The Ras–PI3K Signaling Pathway Is Involved in Clathrin-Independent Endocytosis and the Internalization of Influenza Viruses. Y.Fujioka, M. Tsuda, T. Hattori, J. Sasaki, T. Sasaki, T. Miyazaki & Y. Ohba. PLoS ONE 6(1): e16324 (2011) 



Endocytosis-mediated viral entry

Influenza viruses internalize into host cells via calcium signaling

A variety of viruses are known to enter host cells via endocytosis. Although the specific endocytic pathway for internalization of each virus has been demonstrated, it is also known to be redundant and remains controversial. For instance, Influenza viruses are reported to be internalized into host cells via clathrin-dependent endocytosis. Nevertheless, inhibition of this pathway failed to interfere influenza virus infection.
We have recently reported that influenza viruses activate Ras-PI3K signaling to expedite their efficient incorporation into cells via clathrin-independent endocytosis. We also revealed that the Ras-PI3K activation was preceded by an increase in intracellular calcium concentration (Figure 2). Interestingly, the calcium elevation activates not only Ras-PI3K signaling but also a variety of signaling networks, contributing to the recruitment of various types of endocytosis during the infection (Figure 3). These results together disclosed intracellular signaling networks activated upon influenza virus infection, which are required for the incorporation into cells via endocytosis.






【Related papers】

  • The Ras–PI3K Signaling Pathway Is Involved in Clathrin-Independent Endocytosis and the Internalization of Influenza Viruses. Y.Fujioka, M. Tsuda, T. Hattori, J. Sasaki, T. Sasaki, T. Miyazaki & Y. Ohba. PLoS ONE 6(1): e16324 (2011)
  • A Ca2+-dependent signalling circuit regulates influenza A virus internalisation and infection. Y. Fujioka, M. Tsuda, A. Nanbo, T. Hattori, J. Sasaki, T. Sasaki, T. Miyazaki, and Y. Ohba. Nat. Commun. 4: 2763 (2013)


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Ebora viruses internalize into host cells via macropinocytosis

Ebolavirus is a filamentous, enveloped, non-segmented negative-sense RNA virus that belongs to the family Filoviridae (Fig.1). The Ebolavirus infection causes a 50-90% lethal hemorrhagic fever in humans and nonhuman primates. Because of its high pathogenicity, its potential use as a bioweapon, and the current lack of vaccines and antivirals to combat Ebolavirus infection, Bio Safety Level (BSL)-4 containment is required in studies with live Ebolavirus

Viruses hijack a variety of cellular endocytic pathways to internalize into the host cells. Although Ebolavirus is believed to internalize into the target cells via endocytic pathway(s), the mechanism of entry of Ebolavirus remains poorly understood.

To elucidate the mechanism of Ebolavirus entry, we established a real-time monitoring system of fluorescent-labeled biologically contained Ebolavirus virions and viral-like particles, which retain the morphologies of the authentic Ebolavirus virions, in live cells (Fig.1).

Our real-time imaging system revealed that Ebolavirus s virion is likely internalized via macropinocytosis and its entry is a viral spike protein, Glycoprotein (GP)-dependent (Fig.2). Macropinocytosis is an active in dendritic cells and macrophages stimulated by growth factors, which are known as the initial targets of Ebolavirus infection. Induction of macropinocytosis yields to the formation of specific endosomes (macropinosomes), which are relatively large that possess the enough capacity to uptake Ebolavirus virions.

 The present study provides new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection.

Real-time monitoring of Ebolavirus entry.en

Fig.1 Real-time monitoring of Ebolavirus entry
Fluorescentlly labeled Ebolavirus particles were absorbed to cell surfaces for 30 min on ice. The cells were incubated at 37°C and time-lapse images were acquired using a confocal laser scanning macroscope (left). Still frames at indicated time (seconds) after the temperature-shift to 37°C are shown. Co-locallized virions (red) with macropinosomes (green) are indicated with arrows (right bottom). 


Hypothetical model of GP- and size-dependent .en


Fig.2  Hypothetical model of GP- and size-dependent viral entry.
For Ebolavirus entry, the binding of GP spiked on Ebolavirus virions to the cellular receptor(s) (1) initiates the activation of signal transduction pathways followed by the actin-dependent membrane ruffling (2). The virions are internalized via macriounocytosis (3). Macropinosomes containing the virions are eventually fused to endosomes (late maturation), resulting in the fusion of the viral envelop and endosomal membrane (4). 



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Signal transduction network between heterologous cells

Epstein–Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. EBV has been implicated as a cause of lymphomas and epithelial malignancies. We are studying to clarify the possible roles of cell-to-cell communication in EBV-associated malignancies. 


1. Roles of exosomes derived from Epstein-Barr virus-infected cells in EBV-associated malignancies.

Exosomes are microvesicles actively secreted into all body fluids from various cell types. Exosomes play important roles in adaptive immune responses to pathogens and tumors by transferring proteins, soluble factors, mRNA, and microRNAs to the recipient cells. We first showed that exosomes released from EBV-infected B cells up-regulated cell proliferation and the expression of adhesion molecules in the recipient epithelial cells. Currently we are investigating the possible roles of exosomes derived from EBV-infected B cells in EBV-associated malignancies. We are also examining the possibility of the exosomes as potential biomarkers in diagnosis of EBV-associated malignancies.


2. Characterization of cell-to-cell contact-mediated EBV transmission

Previous observations demonstrate that infection of epithelial cells with EBV is predominately mediated by cell-to-cell contact with EBV-infected B cells. Recently we observed that cell-to-cell contact induces multiple cell signaling pathways in both EBV-infected B cells and epithelial cells, contributing to the induction of the viral lytic cycle in B cells and the enhancement of viral transmission to epithelial cells. Currently we are investigating our hypothetical models; exosomes may stabilize cell-to-cell contact between EBV-infected cells and epithelial cells by transferring adherent molecules, and facilitate EBV infection into target epithelial cells.

Hypothetical model of cell-to-cell contact-mediated EBV transmission.en


Fig1. Hypothetical model of cell-to-cell contact-mediated EBV transmission 


【Related papers】

  •  Nanbo A, Terada H, Kachi K, Takada K, Matsuda T : Roles of Cell Signaling Pathways in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission, Journal of Virology, 86, 9285-9296 (2012)
  • Iizasa H, Nanbo A, Nishikawa J, Yoshiyama H:EBV-associated Gastric Carcinoma, Viruses,  4, 3420-3439 (2012)

  • Nanbo A, Kawanishi E, Yoshida R, Yoshiyama H : Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveolae-dependent endocytosis and promote phenotypic modulation in the target cells. Journal of Virology, 87, 10334-1034 (2013)



Signal transduction of biological rhythm

Circadian rhythm is a crucial factor in regulating a wide range of physiological functions such as the endocrine system and the sleep-wake cycle. The systemic circadian system could be broken down into cellular rhythms, which are maintained by periodic expression of a set of clock genes and proteins. Transcription of clock genes is accelerated by the specific transcription factors, activities of which are suppressed by their own gene products, thereby making a negative feedback loop. The center for circadian system in mammals resides in the suprachiasmatic nucleus of hypothalamus (SCN). Rhythms in the SCN are synchronized to external day-night cycle via retina, and peripheral clocks are entrained by signal(s) from the SCN. Recent studies have shown circadian rhythms of intracellular proteins not only in their amounts but also at the level of posttranslational modification. We currently try to both qualitatively and quantitatively observe circadian rhythms of protein status, including subcellular localization, posttranslational modification, and interaction with other proteins, by means of fluorescence bioimaging.