New Regulators of Clathrin-Mediated Endocytosis Identified in <i>Saccharomyces cerevisiae</i> by Systematic Quantitative Fluorescence Microscopy

Farrell, Kristen B.; Grossman, Caitlin; Pietro, Santiago M. Di

doi:10.1534/genetics.115.180729

Cited by 10 publications

(27 citation statements)

References 39 publications

(62 reference statements)

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“…Like TDP-43 [26], expression of FUS also impaired endocytosis rates, evident by reduced vacuolar staining over the same time period (Fig 4B-D). Consistent with previous mammalian [31,33] and yeast cell findings [46], inactivation of Cdc48 also impaired endocytosis rates to a similar extent as FUS expression, though not quite as strikingly as TDP-43 expression ( Fig. 4A-D).…”

Section: Endocytosis Rate Is Regulated By Cdc48 Tdp-43 and Fussupporting

confidence: 91%

“…Several pieces of evidence suggest that Cdc48 and VCP's effect on TDP-43 and FUS toxicity and turnover may depend on an endocytosis-promoting function. First, inactivation of Cdc48 [46], VCP [33], or expression of either FUS or TDP-43 impair endocytosis ( Fig. 4-5).…”

Section: Discussionmentioning

confidence: 99%

“…First, partial and strong inhibition of Cdc48 at 30°C and 35°C respectively enhanced FUS toxicity in the cdc48-3 strain ( Fig S2A). Second, inactivation of Cdc48 at 35°C also lead to a significant increase in FUS protein stability ( Fig S2B) Third, of all Ubx null strains examined, only deletion of UBX3, which functions in endocytosis [46], clearly increased FUS toxicity ( Fig S2C). Interestingly, as with TDP-43, bre5Δ and ubp3Δ strains also suppressed FUS toxicity ( Fig S2D).…”

Section: Fus Toxicity and Turnover Depend On Endocytic Function And Cmentioning

confidence: 94%

“…2A). Importantly, only deletion of UBX3, which functions along with Cdc48 in yeast endocytosis [46], clearly exhibited enhanced TDP-43 toxicity ( Fig. 2A).…”

Section: Cdc48 Regulates Tdp-43 Toxicity In Part By Endocytosismentioning

confidence: 99%

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Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

Liu¹,

Byrd²,

Pei³

et al. 2019

Preprint

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Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mis-localize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mis-localization and aggregation are implicated in ALS pathology, though the mechanisms of TDP-43 and FUS cytoplasmic toxicity remains unclear.Recently, we determined that the endocytic function aids turnover of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 co-factor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in S. cerevisiae. Cdc48 physically interacts and co-localizes with TDP-43, as does VCP in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function, but not autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identifies a role for Cdc48/VCP and endocytosis function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise. KeywordsCdc48, VCP, Endocytosis, TDP-43, FUS, ALS Abbreviations ALS -Amyotrophic Lateral Sclerosis CORVET -class C core vacuole/endosome tethering CHX -Cycloheximide EGFR -Epidermal growth factor receptor FTLD -Frontotemporal lobar dementia FUS -Fused in Sarcoma HOPS -homotypic fusion and vacuole protein sorting IBMPFD -Inclusion Body Myopathy with early onset Pagets disease and Frontotemporal Dementia RRM -RNA Recognition Motif SG -Stress granule TDP-43 -TAR DNA binding protein 43 VCP -Vasolin containing proteinUnder conditions of cellular stress, both TDP-43 and FUS re-localize into cytoplasmic stress granules (SGs) [6,7], which are dynamic mRNA-protein assemblies implicated in regulating mRNA function [8,9]. Given this, and in light of ALS-associated mutations that generally reduce SG dynamics, SGs have been theorized to promote formation of TDP-43 and FUS aggregates that are observed in ALS patients [10,11], though recent studies suggest SG-independent aggregation mechanism also likely exist [12][13] [14]. Regardless, various studies indicate that TDP-43 and FUS aggregates, or simply excessive cytoplasmic localization of TDP-43 or FUS, may result in a toxic gain of function that leads to motor-neuron degeneration [15][16][17][18][19] [20,21]. While the mechanism of such toxicity remains unclear, preventing aggregation and/or enhancing clearance of cytoplasmic TDP-43 and FUS is of considerable therapeutic interest. This approach has shown promise in various ALS models where preventing SG assembly or upregulating cytoplasmic protein turnover has been tested [22][23][24][25][26].Recently, we and others demonstrated that under normal growth conditions, TDP-43 toxicity, aggregation and prote...

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Section: Endocytosis Rate Is Regulated By Cdc48 Tdp-43 and Fussupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Fus Toxicity and Turnover Depend On Endocytic Function And Cmentioning

confidence: 94%

“…2A). Importantly, only deletion of UBX3, which functions along with Cdc48 in yeast endocytosis [46], clearly exhibited enhanced TDP-43 toxicity ( Fig. 2A).…”

Section: Cdc48 Regulates Tdp-43 Toxicity In Part By Endocytosismentioning

confidence: 99%

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Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

Liu¹,

Byrd²,

Pei³

et al. 2019

Preprint

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“…In addition, the ubiquitin regulatory X (UBX)-domaincontaining protein Ubx3 regulates the rate of Ede1 recruitment to endocytic sites (see poster). This regulation is proposed to be mediated by an interaction between Ubx3 and Cdc48, an AAA-ATPase ubiquitin-editing complex (Farrell et al, 2015). These observations suggest that dynamic ubiquitylation and deubiquitylation are important for regulating the role of Ede1 in CME site initiation and maturation.…”

Section: Ubiquitylationmentioning

confidence: 93%

Clathrin-mediated endocytosis in budding yeast at a glance

Drubin

Sun

2016

Journal of Cell Science

106

105

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Clathrin-mediated endocytosis is an essential cellular process that involves the concerted assembly and disassembly of many different proteins at the plasma membrane. In yeast, live-cell imaging has shown that the spatiotemporal dynamics of these proteins is highly stereotypical. Recent work has focused on determining how the timing and functions of endocytic proteins are regulated. In this Cell Science at a Glance article and accompanying poster, we review our current knowledge of the timeline of endocytic site maturation and discuss recent works focusing on how phosphorylation, ubiquitylation and lipids regulate various aspects of the process.

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Phenomics approaches to understand genetic networks and gene function in yeast

Yeung

Sahin

Andrews

2022

Biochemical Society Transactions

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Over the past decade, major efforts have been made to systematically survey the characteristics or phenotypes associated with genetic variation in a variety of model systems. These so-called phenomics projects involve the measurement of ‘phenomes’, or the set of phenotypic information that describes an organism or cell, in various genetic contexts or states, and in response to external factors, such as environmental signals. Our understanding of the phenome of an organism depends on the availability of reagents that enable systematic evaluation of the spectrum of possible phenotypic variation and the types of measurements that can be taken. Here, we highlight phenomics studies that use the budding yeast, a pioneer model organism for functional genomics research. We focus on genetic perturbation screens designed to explore genetic interactions, using a variety of phenotypic read-outs, from cell growth to subcellular morphology.

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New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Cited by 10 publications

References 39 publications

Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

Clathrin-mediated endocytosis in budding yeast at a glance

Phenomics approaches to understand genetic networks and gene function in yeast

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