Biology subjects

Ijaz, F.

Publications and source records attributed to Ijaz, F..

5 recordsLinked to original sources

bioRxiv2025

Enhanced release of ciliary extracellular vesicles suppresses cell migration and promotes cell aggregation

A primary cilium is a hair-like organelle that protrudes from the cell surface in many cell types. Growing evidence indicates that extracellular vesicles are released from primary cilia, and research is increasingly focused on defining the functions of these cilia-derived extracellular vesicles (EVs). EVs are known to modulate the behavior of various cancer cells, and structural and functional abnormalities in primary cilia have been reported in multiple cancer types. We previously demonstrated that PANC-1 cells, a human pancreatic ductal adenocarcinoma cell line, acquire enhanced primary cilia formation after surviving solitary culture conditions, and that their cilia contribute to tumor-like cell mass formation. Here, we explored part of the underlying mechanism of this phenotype by investigating the contribution of EVs released from the primary cilia of PANC-1 cells. PANC-1 clones generated by limiting dilution exhibited enhanced ciliogenesis and distinct ciliary morphologies compared with parental cells. These clones also released higher levels of cilia-derived EVs, including an expanded population of freely floating EVs within the culture environment. Biochemical analyses further showed that this increase was selective for primary cilia-derived EVs rather than reflecting a global rise in total EV production. Functionally, EV fractions enriched in cilia-derived EVs suppressed parental PANC-1 cell migration, altered cell morphology, and promoted cell aggregation, mimicking key behavioral traits of solitary condition-surviving PANC-1 clones. Together, these findings identify enhanced release of primary cilia-derived EVs as a distinct feature of PANC-1 cells adapted to solitary growth and suggest their potential involvement in the malignant and metastatic behaviors of pancreatic cancer.

cell biology↗

bioRxiv2025

Primary Cilium-dependent Humoral Bioactive Factors Acts in a Paracrine Manner to Control Fibroblast Cell Migration

The primary cilium is classically recognized as a signal-reception hub, yet its role in mediating cell-to-cell communication via signal spread remains poorly defined. Here, we uncover a previously unrecognized paracrine function of the primary cilium in fibroblasts. Conditioned medium from ciliated wild-type NIH/3T3 cells enhanced wound healing in primary cilium-deficient fibroblasts, in contrast to conditioned medium derived from primary cilium-deficient NIH/3T3-Kif3a-KO or NIH/3T3-Dync2h1-KO cells. Fractionation of conditioned medium revealed that the wound healing activity resided predominantly in the 100K x g soluble supernatant (Sup-100K), rather than in extracellular vesicle (EV) fractions. Untargeted metabolomic analysis identified lysophosphatidylcholine (LPC) (14:0) as a key bioactive metabolite enriched in WT-Sup-100K secretome. Supplementation of LPC(14:0) restored wound healing capacity in NIH/3T3-Kif3a-KO cells to levels comparable to WT-Sup-100K treatment. Transcriptomic profiling of target cells revealed that WT-Sup-100K upregulated expression of extracellular matrix (ECM)-associated genes, including Ogn, Igf2, and Mfap4, while EVs modestly enhanced early ECM remodeling via induction of Nid2. Together, these findings demonstrate that the primary cilium coordinates a wound healing secretome in fibroblasts through the regulated release of LPC(14:0) and other soluble factors that activate ECM-remodeling pathways in recipient cells. This work expands the functional repertoire of the primary cilium and establishes its critical role in coordinating paracrine regenerative signaling.

cell biology↗

bioRxiv2024

Excessive F-Actin and Microtubule Formation Mediates Primary Cilia Shortening and Loss in Response to Increased Extracellular Osmotic Pressure

The primary cilium is a small organelle protruding from the cell surface and is recognized as an antenna for signals from the extracellular milieu. Maintenance of primary cilia structure is crucial for proper behaviors of cells, tissues, and organs. While a dozen of studies have reported that several genetic factors impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock with increased extracellular sodium chloride concentration induced shortenings and disassembly of primary cilia in murine intramedullary collecting ducts cells. The shortening of primary cilia caused by hyperosmotic shock followed a loss of axonemal microtubules and delocalization of pericentriolar materials (PCMs). The primary cilia shortening/disassembly and PCMs delocalization were reversible. In parallel with these hyperosmotic shock-induced changes of primary cilia and PCMs, excessive microtubule and F-actin formation occurred in the cytoplasm. A microtubule-disrupting agent, Nocodazole, prevented the hyperosmotic shock-induced primary cilia disassembly partially, while preventing the delocalization of PCMs almost 100%. An actin polymerization inhibitor, Latrunculin A, also prevented partially the hyperosmotic shock-induced primary cilia shortening and disassembly, while preventing the delocalization of PCMs almost 100%. Taken together, we demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and PCMs in a manner dependent on excessive formation of microtubule and F-actin.

cell biology↗

bioRxiv2022

A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using "face-to-face" primers where the 3 ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. We demonstrate six examples of CRISPR/Cas9-engineered knockout cells, to screen indels to obtain potential KO cell clones utilizing our approach. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. Thus, DST-PCR coupled with TBE-High-Resolution PAGE is a simple method for genotyping of the CRISPR/Cas9-engineered cell lines, which can be performed without special equipment and techniques.

cell biology↗

bioRxiv2020

Knock-in of labeled proteins into 5'UTR enables highly efficient generation of stable cell lines

Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biological technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5UTR of the endogenous gene loci. With this 5UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high knock-in efficiencies ranging from 50 to 80%. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5UTR is a hotspot for targeted insertion of gene sequences and allows the stable expression of tagged proteins from endogenous loci in mammalian cells.

cell biology↗