Biology subjects

Gollapudi, S.

Publications and source records attributed to Gollapudi, S..

5 recordsLinked to original sources

bioRxiv2023

Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple groups have reported that early endocytic proteins form flexible condensates, which provide a platform for efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic assemblies. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live cell settings, dynamic exchange of Eps15 proteins, a hallmark of liquid-like systems, was modulated by Eps15-Ub interactions. These results collectively suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic recycling. Significance StatementThe assembly of proteins into dynamic, liquid-like condensates is an emerging principle of cellular organization. During clathrin-mediated endocytosis, a liquid-like protein network catalyzes vesicle assembly. How do cells regulate these assemblies? Here we show that ubiquitin and endocytic proteins form a dynamic, mutually-reinforcing protein network in vitro and in live cells. To probe the impact of ubiquitylation on the dynamics of endocytosis, we engineered opto-genetic control over recruitment of proteins to nascent endocytic sites. While recruitment of wildtype proteins promoted endocytosis, recruitment of deubiquitylases, enzymes capable of removing ubiquitin, resulted in disassembly of endocytic sites within minutes. These results illustrate that ubiquitylation can regulate the fate of endocytic structures, elucidating a functional connection between protein condensates, endocytosis, and ubiquitin signaling.

cell biology↗

bioRxiv2023

Selective endocytic uptake of targeted liposomes occurs within a narrow range of liposome diameter

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays, rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we have employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes, rather than the density of the ligands on their surfaces, primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

bioengineering↗

bioRxiv2023

Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited via inotropic effectors

Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. It remains unresolved whether mavacamten permanently sequesters these myosin heads in the off state(s) or whether these heads can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by Ca2+, increased heart rate, stretch, and {beta}-adrenergic ({beta}-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that, in presence of mavacamten, Ca2+ increases myosin ATPase activity by shifting myosin heads from the reserve super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca2+ and passive lengthening can shift ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments in the presence of mavacamten. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that {beta}-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, at least partially, thus leading to preservation of cardiac reserve mechanisms. Significance statementMavacamten is the first myosin-targeted small molecule inhibitor approved by the FDA to treat obstructive hypertrophic cardiomyopathy by attenuating myocardial hyperdynamic contraction. The recruitment of cardiac contractility is, however, vital to ensure sufficient cardiac output during increased physiological demand. Here we show that major inotropic effectors are at least partially preserved in the setting of mavacamten, resulting in maintenance of cardiac reserve mechanisms. These results not only suggest an alternative mechanistic explanation, beyond mere LV outflow tract obstruction removal, for the clinically observed increase in peak oxygen uptake with exercise in HCM patients receiving mavacamten, but also lay the groundwork for a potential methodology to investigate the sarcomeric basis of chronotropic incompetence in disease states to motivate new therapeutic interventions.

physiology↗

bioRxiv2021

A cancer-associated RNA polymerase III identity drives robust transcription and expression of SNAR-A noncoding RNA

Dysregulation of the RNA polymerase III (Pol III) transcription program, which synthesizes tRNA and other classes of small noncoding RNA critical for cell growth and proliferation, is associated with cancer and human disease. Previous studies have identified two distinct Pol III isoforms defined by the incorporation of either subunit POLR3G (RPC7) during early development, or POLR3GL (RPC7{beta}) in specialized tissues. Though POLR3G is re-established in cancer and immortalized cell lines, the contributions of these isoforms to transcription potential and transcription dysregulation in cancer remain poorly defined. Using an integrated Pol III genomic profiling approach in combination with in vitro differentiation and subunit disruption experiments, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. In particular, we observe that a specific class of small noncoding RNA, SNAR-A, is exquisitely sensitive to the availability of subunit POLR3G in proliferating cells. Taken further, large-scale analysis of Pol III subunit expression and downstream chromatin features identifies concomitant loss of POLR3G and SNAR-A activity across a multitude of differentiated primary immune cell lineages, and conversely, coordinate re-establishment of POLR3G expression and SNAR-A features in a variety of human cancers. These results altogether argue against strict redundancy models for subunits POLR3G and POLR3GL, and instead support a model in which Pol III identity itself functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.

molecular biology↗

bioRxiv2020

CONSERVED INTERACTIONS IN CARDIAC SYNTHETIC THICK FILAMENTS DIFFERENTLY AFFECT MYOSIN SUPER-RELAXED STATE IN HEALTHY, DISEASE AND MAVACAMTEN-TREATED MODELS

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low ionic strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filament. While these STFs have been extensively used for structural characterization, their use for functional studies has been very limited. In this report, we show that the ultra-low ATP-consuming super-relaxed (SRX) state of myosin is electrostatically more stable in STFs as compared with shorter myosin sub-fragments that lack the distal tail required for thick filament assembly. However, this electrostatic stability of the SRX state is weakened by phosphorylation of myosin light chains or the hypertrophic cardiomyopathy-causing myosin R403Q mutation. We also show that ADP binding to myosin depopulates the SRX population in STFs made of wild-type (WT) myosin, but not in S1, HMM, or STFs made of mutant R403Q myosin. Collectively, these findings emphasize that a critical network of inter- and intra-molecular interactions that underlie the SRX state of myosin are mostly preserved in STFs, establishing it as a native-like tool to interrogate myosin regulation. Next, using STFs, we show that a clinical-stage small molecule inhibitor, mavacamten, is more effective in promoting the myosin SRX state in STFs than in S1 or HMM and that it is equally potent in STFs made of atrial-WT, ventricular-WT, and mutant-R403Q myosin. Also, we found that mavacamten-bound heads are not permanently protected in the SRX state but can be recruited in response to physiological perturbations, thus providing new insights into its inhibitory mechanism.

biochemistry↗