dictyNews
Electronic Edition
Volume 49, number 18
July 14, 2023
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Abstracts
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A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on
macropinosomes and phagosomes
James H. Vines1, Hannes Maib2, Catherine M. Buckley1, Aurelie
Gueho3, Zhou Zhu1, Thierry Soldati3, David H. Murray2 and
Jason S. King1*
1School of Biosciences, University of Sheffield, Firth Court
Western Bank, Sheffield, UK.
2Division of Molecular, Cell and Developmental Biology, School
of Life Sciences, University of Dundee.
3Department of Biochemistry, Faculty of Science, University of
Geneva, Switzerland.
*Corresponding author: Jason King
Journal of Cell Biology, (2023) 222 (9): e202209077
https://rupress.org/jcb/article/222/9/e202209077/214199/
A-PI-3-5-P2-reporter-reveals-PIKfyve-activity-and
Phosphoinositide signalling lipids (PIPs) are key regulators
of membrane identity and trafficking. Of these, PI(3,5)P2 is
one of the least well understood, despite key roles in many
endocytic pathways including phagocytosis and macropinocytosis.
PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve,
which is critical for phagosomal digestion and antimicrobial
activity. However PI(3,5)P2 dynamics and regulation remain
unclear due to lack of reliable reporters. Using the amoeba
Dictyostelium discoideum we identify SnxA as a highly-selective
PI(3,5)P2-binding protein and characterise its use as a reporter
for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using
GFP-SnxA we demonstrate that Dictyostelium phagosomes and
macropinosomes accumulate PI(3,5)P2 three minutes after
engulfment but is then retained differently, indicating pathway-
specific regulation. We further find that PIKfyve recruitment
and activity are separable, and that PIKfyve activation
stimulates its own dissociation. SnxA is therefore a new tool
for reporting PI(3,5)P2 in live cells that reveals key
mechanistic details of the role and regulation of
PIKfyve/PI(3,5)P2.
Submitted by Jason King [[log in to unmask]]
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ER-dependent membrane repair of mycobacteria-induced vacuole
damage
Aby Anand 1, Anna-Carina Mazur 1, Patricia Rosell-Arevalo 2, Rico
Franzkoch 3, Leonhard Breitsprecher 3, Stevanus A. Listian 1,
Sylvana V. Hüttel 1, Danica Müller 1, Deise G. Schäfer 1, Simone
Vormittag 4, Hubert Hilbi 4, Markus Maniak 5, Maximiliano G.
Gutierrez 2 and Caroline Barisch 1,6,7,8,*
11 Division of Molecular Infection Biology, Department of Biology &
Center of Cellular Nanoanalytics, University of Osnabrück, Osnabrück,
Germany
2 Host–Pathogen Interactions in Tuberculosis Laboratory, The Francis
Crick Institute, London, United Kingdom
3 iBiOs–integrated Bioimaging Facility, Center of Cellular
Nanoanalytics, University of Osnabrück, Osnabrück, Germany
4 Institute of Medical Microbiology, University of Zürich, Zürich,
Switzerland
5 Department of Cell Biology, University of Kassel, Kassel, Germany
6 Centre for Structural Systems Biology, Hamburg, Germany
7 Research Center Borstel, Leibniz Lung Center, Sülfeld, Germany
8 Biology Department, University of Hamburg, Hamburg, Germany
mbio, in press
Several intracellular pathogens, such as Mycobacterium tuberculosis,
damage endomembranes to access the cytosol and subvert innate
immune responses. The host counteracts endomembrane damage by
recruiting repair machineries that retain the pathogen inside the vacuole.
Here, we show that the endoplasmic reticulum (ER)-Golgi protein
oxysterol binding protein (OSBP) and its Dictyostelium discoideum
homologue OSBP8 are recruited to the Mycobacterium-containing
vacuole (MCV) dependent on the presence of the ESX-1 secretion
system, suggesting that their mobilization is associated with membrane
damage. Lack of OSBP8 causes a hyperaccumulation of
phosphatidylinositol-4-phosphate (PI4P) on the MCV and decreased
cell viability. OSBP8-depleted cells had reduced lysosomal and
degradative capabilities of their vacuoles that favoured mycobacterial
growth. In agreement with a potential role of OSBP8 in membrane repair,
human macrophages infected with M. tuberculosis recruited OSBP in an
ESX-1-dependent manner. These findings identified an ER-dependent
repair mechanism for restoring MCVs in which OSBP8 functions to
equilibrate PI4P levels on damaged membranes.
Submitted by Caroline Barisch [[log in to unmask]]
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