Biology subjects

Rahi, A.

Publications and source records attributed to Rahi, A..

5 recordsLinked to original sources

bioRxiv2024

The Hexosamine Biosynthetic Pathway alters the cytoskeleton to modulate cell proliferation and migration in metastatic prostate cancer

Castration-resistant prostate cancer (CRPC) progresses despite androgen deprivation therapy, as cancer cells adapt to grow without testosterone, becoming more aggressive and prone to metastasis. CRPC biology complicates the development of effective therapies, posing challenges for patient care. Recent gene-expression and metabolomics studies highlight the Hexosamine Biosynthetic Pathway (HBP) as a critical player, with key components like GNPNAT1 and UAP1 being downregulated in metastatic CRPC. GNPNAT1 knockdown has been shown to increase cell proliferation and metastasis in CRPC cell lines, though the mechanisms remain unclear. To investigate the cellular basis of these CRPC phenotypes, we generated a CRISPR-Cas9 knockout model of GNPNAT1 in 22Rv1 CRPC cells, analyzing its impact on metabolomic, glycoproteomic, and transcriptomic profiles of cells. We hypothesize that HBP inhibition disrupts the cytoskeleton, altering mitotic progression and promoting uncontrolled growth. GNPNAT1 KO cells showed reduced levels of cytoskeletal filaments, such as actin and microtubules, leading to cell structure disorganization and chromosomal mis-segregation. GNPNAT1 inhibition also activated PI3K/AKT signaling, promoting proliferation, and impaired cell adhesion by mislocalizing EphB6, enhancing migration via the RhoA pathway and promoting epithelial-to-mesenchymal transition. These findings suggest that HBP plays a critical role in regulating CRPC cell behavior, and targeting this pathway could provide a novel therapeutic approach.

cancer biology↗

bioRxiv2024

SAXS/MC studies of the mixed-folded protein Cdt1 reveal monomeric, folded over conformations

Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, "moonlighting" role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the protein in silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent "folded over" conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.

biophysics↗

bioRxiv2023

Nuclear lamin A-associated proteins are required for centromere assembly

Many Lamin A-associated proteins (LAAPs) that are key constituents of the nuclear envelope (NE), assemble at the "core" domains of chromosomes during NE reformation and mitotic exit. However, the identity and function of the chromosomal core domains remain ill-defined. Here, we show that a distinct section of the core domain overlaps with the centromeres/kinetochores of chromosomes during mitotic telophase. The core domain can thus be demarcated into a kinetochore proximal core (KPC) on one side of the segregated chromosomes and the kinetochore distal core (KDC) on the opposite side, close to the central spindle. We next tested if centromere assembly is connected to NE re-formation. We find that centromere assembly is markedly perturbed after inhibiting the function of LMNA and the core-localized LAAPs, BANF1 and Emerin. We also find that the LAAPs exhibit multiple biochemical interactions with the centromere and inner kinetochore proteins. Consistent with this, normal mitotic progression and chromosome segregation was severely impeded after inhibiting LAAP function. Intriguingly, the inhibition of centromere function also interferes with the assembly of LAAP components at the core domain, suggesting a mutual dependence of LAAP and centromeres for their assembly at the core domains. Finally, we find that the localization of key proteins involved in the centromeric loading of CENP-A, including the Mis18 complex and HJURP were markedly affected in LAAP-inhibited cells. Our evidence points to a model where LAAP assembly at the core domain serves a key function in loading new copies of centromeric proteins during or immediately after mitotic exit.

cell biology↗

bioRxiv2022

The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit

The Ndc80 kinetochore complex is essential for robust kinetochore-microtubule (k-MT) attachments during mitosis. Ndc80 has been shown to recruit the Ska1 complex to kinetochores, where Ska1 is thought to aid in k-MT coupling by Ndc80. Our previous work has shown that Cdt1, a DNA replication licensing factor, is a novel mitotic spindle-associated protein that is also recruited to kinetochores via Ndc80 and is required for stabilizing k-MT attachments. In this study, we developed auxin-induced degron (AID)-tagging to validate the previously demonstrated mitotic role of Cdt1. We demonstrate a direct interaction between Cdt1 and Ska1 that is essential for proper recruitment of Cdt1 to kinetochores and spindle microtubules. We find that Cdk1dependent phosphorylation of Cdt1 during mitosis is critical for Ska1-binding, consequently regulating the stabilization of metaphase k-MT attachments and normal mitotic progression. Total internal reflection fluorescence microscopy (TIR-FM) experiments reveal that Cdt1 synergizes with the Ndc80 and the Ska1 complexes for microtubule-binding. Further, we show that single Cdt1 molecules form diffusive tripartite complexes with Ndc80 and Ska1 that can processively track the ends of dynamic microtubules in vitro. Taken together our data identifies a minimal molecular unit responsible for bidirectional processive tip tracking of kinetochores.

cell biology↗

bioRxiv2019

An innovative non-invasive technique for subcutaneous tumour measurements

In oncological drug development, animal studies continue to play a central role in which the volume of subcutaneous tumours is monitored to assess the efficacy of new drugs. Tumour volume is currently estimated by measuring length and width with callipers and then estimating the volume of the tumour as if it were a regular spheroid. However, this method is subjective, insufficiently traceable, and is subject to error in the accuracy of volume estimates as tumours frequently are irregular.\n\nThis paper explores the extent of inconsistencies in calliper measurements by conducting a statistical review of a large dataset consisting of 2,500 tumour volume measurements from 1,600 mice by multiple operators across 6 mouse strains and 20 tumour models. We also explore the impact of six different tumour morphologies on volume estimation and the detection of treatment effects using a computational tumour growth model. Finally, we propose an alternative method to callipers for estimating volume - BioVolume, a 3D scanning technique. BioVolume simultaneously captures both stereo RGB (Red, Green and Blue) images from different light sources and infrared thermal images of the tumour. It detects the tumour region automatically and estimates the tumour volume in under a second. BioVolume has been tested on a dataset of 297 scans from over 120 mice collected by four different operators.\n\nThis work demonstrates that it is possible to record tumour measurements in a rapid, minimally invasive, morphology-independent way, and with less human-bias compared to callipers, whilst also improving data traceability. Furthermore, the images collected by BioVolume may be useful, for example, as a source of biomarkers for animal welfare and secondary drug toxicity / efficacy.

cancer biology↗