TY - JOUR
T1 - Charge and redox states modulate granulin—TDP-43 coacervation toward phase separation or aggregation
AU - Bhopatkar, Anukool A.
AU - Dhakal, Shailendra
AU - Abernathy, Hannah G.
AU - Morgan, Sarah E.
AU - Rangachari, Vijayaraghavan
N1 - Funding Information:
The authors would like to thank the following agencies for financial support: National Institute on Aging ( 1R56AG062292-01 ) and the National Science Foundation ( NSF CBET 1802793 ) to V.R. The authors also thank the National Center for Research Resources ( 5P20RR01647-11 ) and the National Institute of General Medical Sciences ( 8 P20 GM103476-11 ) from the National Institutes of Health for funding through INBRE for the use of their core facilities, and the National Science Foundation ( NSF MRI 2019023 ) for STED confocal microscope facility. The authors would also like to thank Dr. Jonathan Lindner for his gracious help with the confocal microscope use and troubleshooting.
Publisher Copyright:
© 2022 Biophysical Society
PY - 2022/6/7
Y1 - 2022/6/7
N2 - Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1–7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN's absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.
AB - Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1–7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN's absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.
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U2 - 10.1016/j.bpj.2022.04.034
DO - 10.1016/j.bpj.2022.04.034
M3 - Article
C2 - 35490297
AN - SCOPUS:85130388558
SN - 0006-3495
VL - 121
SP - 2107
EP - 2126
JO - Biophysical Journal
JF - Biophysical Journal
IS - 11
ER -