Biology subjects

Masamsetti, V. P.

Publications and source records attributed to Masamsetti, V. P..

3 recordsLinked to original sources

bioRxiv2018

Nuclear filamentous actin functions in the replication stress response

\"Replication stress\" describes phenomena that alter DNA replication rates 1-3. Multiple architectural challenges within the confined nuclear volume must be navigated during replication to prevent or repair replication stress. Cellular mechanisms potentiating changes in nuclear architecture that facilitate DNA replication remain unclear. Here we show that the ATR, IPMK and mTOR kinases regulate actin polymerisation in human cells to alter nuclear architecture and promote replication fork repair. We demonstrate that replication stress activates mTOR, in an ATR and IPMK-dependent manner, to induce polymerisation of nuclear filamentous actin (F-actin). mTOR and ATR then counteract replication stress-induced nuclear envelope deformation and increase nuclear volume through their regulation of actin dynamics. Additionally, we reveal that FANCD2 labelled replication forks colocalise with actin filaments in late S-phase. mTOR and ATR then regulate the mobility, speed and directionality of stalled replication foci within the three-dimensional nuclear architecture. Importantly, we find nuclear F-actin also acts as a substrate for the directed migration of stalled replication foci to the nuclear periphery. Suppressing mTOR and ATR-dependent actin forces prevents replication fork restart and promotes chromosome segregation errors in primary and cancer cell lines. Together, these data reveal that ATR and mTOR regulate actin dynamics in the replication stress response to alter nuclear architecture and maintain genome stability.

cell biology↗

bioRxiv2018

Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection

Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death promotes genome instability in replication stressed cells through diverse mechanisms depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and genome instability.

molecular biology↗

bioRxiv2018

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.

To investigate how chromatin architecture is spatiotemporally organised at a double strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image correlation spectroscopy (ICS) of histone FLIM-FRET microscopy data acquired in live cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that when coupled with laser micro-irradiation induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ATM and RNF8 regulate rapid chromatin decompaction at DSBs and formation of a compact chromatin ring surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.\n\nSIGNIFICANCE STATEMENTChromatin dynamics play a central role in the DNA damage response (DDR). A long-standing obstacle in the DDR field was the lack of technology capable of visualising chromatin dynamics at double strand break (DSB) sites. Here we describe novel biophysical methods that quantify spatiotemporal chromatin compaction dynamics in living cells. Using these novel tools, we identify how chromatin architecture is reorganised at a DSB locus to enable repair factor access and demarcate the lesion from the surrounding nuclear environment. Further, we identify novel regulatory roles for key DDR enzymes in this process. Finally, we demonstrate method utility with physical, pharmacological and genetic manipulation of the chromatin environment, identifying method potential for use in future studies of chromatin biology.

biophysics↗