July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
IQ-domain GTPase-activating protein1 (IQGAP-1) Modulation of Tight Junction Proteins and Protection against P. aeruginosa Invasion
Author Affiliations & Notes
  • Elizabeth P Shen
    Ophthalmology, Taipei Tzu Chi Hospital, Taipei, Taiwan
  • Fung-Rong Hu
    Ophthalmology, National Taiwan University, Taipei, -- select one --, Taiwan
  • Footnotes
    Commercial Relationships   Elizabeth Shen, None; Fung-Rong Hu, None
  • Footnotes
    Support  NSC-105-2314-B-002-167-MY3
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 504. doi:https://doi.org/
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      Elizabeth P Shen, Fung-Rong Hu; IQ-domain GTPase-activating protein1 (IQGAP-1) Modulation of Tight Junction Proteins and Protection against P. aeruginosa Invasion
      . Invest. Ophthalmol. Vis. Sci. 2018;59(9):504. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To determine the role of IQ-domain GTPase-activating protein1 (IQGAP-1) in tight junction regulation in human corneal epithelial cells (HCECs) and its possible protective effect against P. aeruginosa infection.

Methods : Transformed human corneal epithelial cells (HCECs) and si-IQGAP-1 RNA knockdown HCECs (siHCECs) are used for experimentation. Confocal microscopy is used to locate the proteins actin, zonular occluding-1 (ZO-1), and IQGAP-1 in both HCECs and siHCECs. Transepithelial electrical resistance (TER) is measured and compared between HCECs and siHCECs before and after infection by Type III secretion system (T3SS) invasive genotype P. aeruginosa strain (PAK). Cell viability by trypan blue exclusion and gentamicin invasion assay are performed to compare the relative survival and PAK intracellular invasion between control HCECs and siHCECs.

Results : In control HCECs, IQGAP-1 is located mainly at submembrane intracytoplasmic regions which co-localize mainly with ZO-1 and less with actin. After IQGAP-1 KD, actin and ZO-1 rearrangement is noted with greater co-localization of the two proteins. Confocal z view microscopy further identified the apical positioning of ZO-1 with IQGAP-1 co-localization. Actin is mainly located at lateral and basal cellular borders. IQGAP-1 binds to both actin and ZO-1 at the junction of the apical and lateral cellular region indicating a possible role of IQGAP-1 in modulation of cellular tight junctions. IQGAP-1KD increased the basal TER of HCECs. After PAK infection, IQGAP-1 KD cells could maintain the TER up to 2.5 hrs after infection. Cell viability after PAK infection is also greater for IQGAP-1 KD cells up to 4 hrs after PAK infection. IQGAP-1KD is also protective in preventing PAK intracellular invasion as seen by about 50% decrease in the number of intracellular bacteria.

Conclusions : IQGAP-1 through its binding with actin and ZO-1 may modulate the tight junction of HCECs. IQGAP-1 knockdown may increase the strength of tight junctions and provide a protective effect against P. aeruginosa invasion.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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