The Cdc48 Complex Alleviates the Cytotoxicity of Misfolded Proteins by Regulating Ubiquitin Homeostasis

The accumulation of misfolded proteins is associated with multiple neurodegenerative disorders, but it remains poorly defined how this accumulation causes cytotoxicity. Here, we demonstrate that the Cdc48/p97 segregase machinery drives the clearance of ubiquitinated model misfolded protein Huntingtin (Htt103QP) and limits its aggregation. Nuclear ubiquitin ligase San1 acts upstream of Cdc48 to ubiquitinate Htt103QP. Unexpectedly, deletion of SAN1 and/or its cytosolic counterpart UBR1 rescues the toxicity associated with Cdc48 deficiency, suggesting that ubiquitin depletion, rather than compromised proteolysis of misfolded proteins, causes the growth defect in cells with Cdc48 deficiency. Indeed, Cdc48 deficiency leads to elevated protein ubiquitination levels and decreased free ubiquitin, which depends on San1/Ubr1. Furthermore, enhancing free ubiquitin levels rescues the toxicity in various Cdc48 pathway mutants and restores normal turnover of a known Cdc48-independent substrate. Our work highlights a previously unappreciated function for Cdc48 in ensuring the regeneration of monoubiquitin that is critical for normal cellular function

A 3D+t Laplace operator for temporal mesh sequences

International audienceThe Laplace operator plays a fundamental role in geometry processing. Several discrete versions have been proposed for 3D meshes and point clouds, among others. We define here a discrete Laplace operator for temporally coherent mesh sequences, which allows to process mesh animations in a simple yet efficient way. This operator is a discretization of the Laplace-Beltrami operator using Discrete Exterior Calculus on CW complexes embedded in a four-dimensional space. A parameter is introduced to tune the influence of the motion with respect to the geometry. This enables straightforward generalization of existing Laplacian static mesh processing works to mesh sequences. An application to spacetime editing is provided as example

The absence of specific yeast heat-shock proteins leads to abnormal aggregation and compromised autophagic clearance of mutant Huntingtin proteins

<div><p>The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (<i>HTT</i>) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington’s disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in <i>sse1</i>Δ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance.</p></div

Cells lacking Sse1 and Fes1 exhibit accelerated Htt103QP aggregate formation.

<p>(A) WT, <i>sse1</i>Δ, and <i>fes1</i>Δ cells with <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-GFP</i> plasmid were grown in non-inducible raffinose medium (YEP + Raffinose) to mid log phase, and then galactose was added to induce the overexpression of Flag-Htt103QP-GFP. Cells were collected every hour for microscopy using the EVOS microscope. Representative DIC and fluorescence images are shown for Htt103QP aggregate formation. Scale bar = 5μm. The number of aggregates in each cell were quantified. The percentage of cells with 1, 2 or >2 aggregates (n = 100 cells) is shown. (B) Quantitation of the average number of aggregates per cell in WT, <i>sse1</i>Δ and <i>fes1</i>Δ cells. Averages are from three independent experiments (n > 50 cells). (C) Detection of Htt103QP protein aggregates using agarose gel electrophoresis. WT and mutant cells were grown in non-inducible raffinose medium (R). After galactose addition, the cells were incubated for 3 hr (G). Protein samples were prepared as described in Materials and Methods and subjected to SDS agarose gel electrophoresis (SDS-AGE) and SDS-PAGE. Two separate WT and <i>sse1</i>Δ strains are shown for reproducibility (left). The results for WT, <i>sse2</i>Δ, <i>fes1</i>Δ, and <i>snl1</i>Δ mutants are shown in the right panel. Pgk1: loading control.</p

Cytosolic Hsp70 nucleotide exchange factors Sse1 and Fes1 are required for Htt103QP inclusion body (IB) formation.

<p>(A) Htt103QP overexpression and the growth of WT and <i>sse1</i>Δ cells. Cells containing <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-GFP</i> were grown to saturation, 10-fold diluted, and spotted onto glucose (YPD) or galactose (YEPG) (yeast extract peptone and galactose) plates. The plates were incubated at 30°C for 2 days. (B) Confocal DIC and fluorescent images showing Htt103QP IB formation. WT and <i>sse1</i>Δ cells with <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-GFP</i> were incubated at 30°C in YEPG medium for 16 hrs to induce Htt103QP-GFP expression. The cells were fixed by paraformaldehyde for 5 min and then resuspended in PBS buffer for confocal microscopy. Scale bar = 5μm. <b>(C)</b> 2.5-D analysis showing the intensity of fluorescent GFP peaks of the images from (B). Software used was Zen Blue from Zeiss. (D) Htt103QP IB formation in yeast mutants lacking Hsp70 nucleotide exchange factors (NEFs). Cells with the indicated genotypes were grown and treated as described above and then examined using an EVOS fluorescence microscope. Representative fluorescent images are shown for WT, <i>snl1</i>Δ, <i>sse1</i>Δ, <i>sse2</i>Δ, <i>fes1</i>Δ, and <i>sse1</i>Δ <i>fes1</i>Δ cells expressing Htt103QP-GFP. Scale bar = 5μm. (E) Cells from (D) were quantified for the number of aggregates in each cell: 1, 2 or >2 (n = 100 cells). The results are the average of three independent experiments. (F) The average number of aggregates in WT, <i>sse1</i>Δ, <i>fes1</i>Δ, and <i>sse1</i>Δ <i>fes1</i>Δ cells from (D) were quantified (n > 40 cells). The results are the average of three independent experiments. * indicates statistical comparison between WT and mutants. p < .0001 in all instances.</p

Hsp104-GFP positive aggregates are increased in <i>sse1</i>Δ and <i>ydj1-151</i> mutants.

<p>(A) Hsp104 recruitment to Htt103QP aggregates is independent of chaperones. Cell with indicated genotypes were grown in non-inducible raffinose medium to mid-log phase then galactose was added. Images were taken after incubation for 3 hr. Representative fluorescence images show cellular localization of Hsp104-GFP and Htt103QP-mApple. DIC and merged images are also shown. Scale bar = 5μm. (B) Constitutive appearance of Hsp104-GFP positive aggregates in <i>sse1</i>Δ and <i>ydj1-151</i> mutants. Cells with indicated genotypes were grown in raffinose or galactose containing medium to mid-log phase. Representative fluorescence images are shown. WT cells show predominantly nuclear localized Hsp104-GFP in the absence of Htt103QP overexpression. Scale bar = 5μm. (C) Cells from (B) were quantified based on the presence of Hsp104-GFP positive aggregate(s). Results are the average of three independent experiments (n = 100 cells). All images in this figure was obtained using the EVOS microscope.</p

Hsp40 co-chaperone Ydj1 is required for efficient autophagic degradation of Htt103QP.

<p>(A) Htt103QP autophagy in WT and <i>ydj1-151</i> mutant cells. <i>pep4</i>Δ <i>VPH1-mApple</i> (WT) and <i>ydj1-151 pep4</i>Δ <i>VPH1-mApple</i> (<i>ydj1-151</i>) cells containing <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-GFP</i> were grown in galactose medium (YEPG) at 25°C for 16 hrs. Cells were then shifted to semi-permissive temperatures (32°C) for 10 min prior to glucose and hydroxyurea (HU) addition. Representative fluorescent images are shown for the localization of Htt103QP-GFP and Vph1-mApple (vacuolar marker) before and after addition of glucose and HU. Scale bar = 5μm. (B) Quantitation of cells with Htt103QP IB or aggregates localized outside the vacuole over time. Results from three independent experiments were used for the quantitation. The quantified results were obtained by counting the number of cells containing at least one IB or aggregate (n = 100). (C) The co-localization of Htt103QP and Atg8 in WT and <i>ydj1-151</i> cells. Cells containing <i>GFP-ATG8</i> and <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-mApple</i> were grown in galactose medium YEPG for 16 hrs at 32°C and fixed for microscopy. Representative fluorescence images show the localization of Htt103QP-mApple and GFP-Atg8. (D) Quantitation of total number of Htt103QP aggregates colocalized with GFP-Atg8 in WT and <i>ydj1-151</i> cells (n = 100 IB/aggregates). Quantities indicate the average of three independent experiments. * indicates statistical comparison. p < .005. (E) Quantitation of the number of GFP-Atg8 autophagosomes present in WT and <i>ydj1-151</i> cells incubated in glucose or galactose medium (n = 100 cells). Quantities indicate the average of three independent experiments. All images in this figure were acquired using the EVOS microscope.</p

Sse1 and Fes1 are required for efficient recognition of Htt103QP by autophagy machinery.

<p>(A) The co-localization of Htt103QP and Atg8 in WT, <i>sse1</i>Δ, and <i>fes1</i>Δ cells. Cells containing <i>GFP-ATG8</i> and <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-mApple</i> were grown in galactose medium YEPG for 16 hrs and fixed for microscopy. Representative fluorescence images show the localization of Htt103QP-mApple and GFP-Atg8. Scale bar = 5μm. (B) Quantitation of total number of Htt103QP aggregates colocalized with GFP-Atg8 in WT, <i>sse1</i>Δ, and <i>fes1</i>Δ cells (n = 100 IB/aggregates). Quantities indicate the average of three independent experiments. * indicates statistical comparison. p < .001. (C) Quantitation of the number of GFP-Atg8 autophagosomes present in WT, <i>sse1</i>Δ and <i>fes1</i>Δ cells incubated in glucose or galactose medium (n = 100 cells). Quantities indicate the average of three independent experiments. (D) Sse1 and Fes1 are not required for starvation-induced autophagy. WT, <i>sse1</i>Δ, and <i>fes1</i>Δ cells with <i>GFP-ATG8</i> were grown in SC -TRP medium, and then shifted to nitrogen-deficient medium (SD-N) for 24 hr. The cells were collected to detect the presence of GFP-Atg8 and the cleavage product, monomeric GFP, using anti-GFP antibody. Pgk1: loading control. The EVOS microscope was used to obtain the images in this figure.</p

The autophagic degradation of Htt103QP is compromised in <i>sse1</i>Δ and <i>fes1</i>Δ mutants.

<p>(A) The autophagic disposal of Htt103QP in WT and <i>sse1</i>Δ mutant cells. <i>pep4</i>Δ <i>VPH1-mApple</i> (WT) and <i>sse1</i>Δ <i>pep4</i>Δ <i>VPH1-mApple</i> (<i>sse1</i>Δ) cells containing Flag-Htt103QP-GFP were grown in galactose medium (YEPG) at 30°C for 16 hr. Glucose was then added to shut off Htt103QP expression, and 100 mM hydroxyurea (HU) was added to block cell cycle progression. Representative fluorescent images are shown for the localization of Htt103QP-GFP and Vph1-mApple before and after addition of glucose and HU. Vph1-mApple was used to mark vacuole structure. Pep4 is a vacuolar protease, and <i>pep4</i>Δ mutant was used to slow degradation inside the vacuole to enhance visualization. (B) Htt103QP autophagic disposal in WT and <i>fes1</i>Δ mutant cells. A similar protocol was used to examine the vacuolar localization of Htt103QP-GFP. (C and D) Quantitation of cells with Htt103QP IB or aggregates localized outside the vacuole over time. The cells from (A) and (B) are used for the quantitation. The quantified results (three independent repeats) were obtained by counting the number of cells containing at least one IB or aggregate (n = 100). All microscopy in this figure was performed on the EVOS microscope.</p