Report
Cytoplasmic Dynein Is Required for the Spatial Organization of Protein Aggregates in Filamentous Fungi Graphical Abstract
Authors Martin J. Egan, Mark A. McClintock, ..., Hunter L. Elliott, Samara L. Reck-Peterson
Correspondence
[email protected]. edu
In Brief Spatial organization and clearance of damaged proteins is important for cell viability and aging. Egan et al. investigate this process in Aspergillus nidulans, finding that the microtubule-based motor cytoplasmic dynein promotes clearance through coalescence of protein aggregates. This has implications for understanding transport processes in disease.
Highlights d
Aspergillus nidulans protein aggregates coalesce into discrete structures
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Microtubules facilitate the formation of Hsp104-positive inclusions
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Cytoplasmic dynein promotes aggregate clearance through their coalescence
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Impaired aggregate clearance impedes trafficking of conventional dynein cargo
Egan et al., 2015, Cell Reports 11, 201–209 April 14, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.celrep.2015.03.028
Cell Reports
Report Cytoplasmic Dynein Is Required for the Spatial Organization of Protein Aggregates in Filamentous Fungi Martin J. Egan,1,3 Mark A. McClintock,1,3 Ian H.L. Hollyer,1 Hunter L. Elliott,2 and Samara L. Reck-Peterson1,* 1Department
of Cell Biology, Harvard Medical School, Boston, MA 02115, USA and Data Analysis Core, Harvard Medical School, Boston, MA 02115, USA 3Co-first author *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.celrep.2015.03.028 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2Image
SUMMARY
Eukaryotes have evolved multiple strategies for maintaining cellular protein homeostasis. One such mechanism involves neutralization of deleterious protein aggregates via their defined spatial segregation. Here, using the molecular disaggregase Hsp104 as a marker for protein aggregation, we describe the spatial and temporal dynamics of protein aggregates in the filamentous fungus Aspergillus nidulans. Filamentous fungi, such as A. nidulans, are a diverse group of species of major health and economic importance and also serve as model systems for studying highly polarized eukaryotic cells. We find that microtubules promote the formation of Hsp104positive aggregates, which coalesce into discrete subcellular structures in a process dependent on the microtubule-based motor cytoplasmic dynein. Finally, we find that impaired clearance of these inclusions negatively impacts retrograde trafficking of endosomes, a conventional dynein cargo, indicating that microtubule-based transport can be overwhelmed by chronic cellular stress. INTRODUCTION Maintaining the integrity of the cellular proteome is a universal biological challenge that is addressed with a variety of proteostatic mechanisms, including the ubiquitin-proteasome system, autophagy pathways, and molecular disaggregases and chaperones (Tyedmers et al., 2010). Failure of these quality control mechanisms carries severe penalties for the cell, ranging from the cytotoxic accumulation of misfolded and damaged proteins to accelerated cellular aging (Coelho et al., 2013, 2014; Erjavec et al., 2007; Moseley, 2013; Nystro¨m and Liu, 2014; Vendruscolo et al., 2011). Defects in maintaining protein homeostasis are also pervasive in human pathology, particularly in neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (Vendruscolo et al., 2011). Despite such broad consequences, an understanding of the mechanistic relationship
between aberrant protein aggregation and cytotoxicity is still incomplete. Recent studies have focused on how spatial organization of protein aggregates promotes cellular fitness (Moseley, 2013; Sontag et al., 2014; Tyedmers et al., 2010). Coalescence and asymmetric inheritance of proteinaceous inclusions have been described in mammalian cells, budding yeast, fission yeast, and bacteria, and represent a conserved mechanism of cellular aging in which damaged proteins accumulate in mother cells, allowing daughter cells to maximize their replicative potential (Moseley, 2013; Ogrodnik et al., 2014; Sontag et al., 2014; Tyedmers et al., 2010). While the advantages conferred by such spatial quality control are evident across the evolutionary scale, mechanistic details of these defined pathways are still emerging. The role of the cytoskeleton in spatial quality control is an area of active investigation. In fission yeast and bacteria, coalescence and asymmetric inheritance of damaged proteins are independent of cytoskeletal elements (Coelho et al., 2014; Lindner et al., 2008; Winkler et al., 2010). Although there are conflicting reports regarding the dispensability of actin for the coalescence of aggregates into defined inclusions in budding yeast (EscusaToret et al., 2013; Specht et al., 2011; Spokoini et al., 2012), an intact actin cytoskeleton is required for the selective retention of these inclusions in mother cells during cytokinesis (Erjavec et al., 2007; Ganusova et al., 2006; Liu et al., 2010; Tessarz et al., 2009). However, alternative models have been proposed and include the possibility of retention through tethering to organelles (Liu et al., 2010; Spokoini et al., 2012; Zhou et al., 2011, 2014). Microtubules are also involved in spatial quality control. The best studied example is the mammalian aggresome-autophagy pathway, which uses microtubule-based transport to compartmentalize protein aggregates at the microtubule-organizing center (MTOC) during interphase (Chin et al., 2010). Additionally, this association of damaged cellular proteins with the MTOC facilitates their asymmetric inheritance during cytokinesis (Ogrodnik et al., 2014). The contribution of microtubules in other eukaryotes is less clear. Microtubule-dependent inclusions have been reported in yeast, particularly in disease models in which aggregation-prone human proteins localize to aggresome-like structures (Kaganovich et al., 2008; Muchowski et al., 2002; Wang et al., 2009). However, because yeast cells use actin rather Cell Reports 11, 201–209, April 14, 2015 ª2015 The Authors 201
than microtubules for cellular transport, the role of microtubules in forming these inclusions is unclear (Hammer and Sellers, 2012). Here, we sought to understand spatial quality control in filamentous fungi, using Aspergillus nidulans as a model. Filamentous fungi are opportunistic pathogens in humans; pervasive pathogens of important food crops, including corn and rice; and of industrial importance for fermentation, biofuels, and bioremediation (Perez-Nadales et al., 2014). Filamentous fungi colonize substrates through highly polarized, multi-nucleate hyphae, which elongate rapidly at their apices. Unlike budding yeasts, this growth is dependent on the transport of materials along polarized microtubule arrays by molecular motor proteins (Egan et al., 2012a; Steinberg, 2014), making filamentous fungi an excellent model system for studying transport in other polarized cells, such as neurons, in which transport defects are a common pathological feature of neurodegenerative disorders (Encalada and Goldstein, 2014). Using the molecular disaggregase Hsp104 as a marker for global protein aggregation, we found that stress-induced inclusions cluster and coalesce into discrete subcellular structures in a manner dependent on microtubules and the microtubule-based motor dynein. Moreover, we show that impaired clearance of these inclusions can negatively impact the trafficking of cellular cargos, indicating that microtubule-based transport, like other agents of protein quality control, can be overwhelmed by chronic cellular stress. RESULTS Hsp104-Positive Aggregates Form, Cluster, and Coalesce during Recovery from Transient Heat Stress Hsp104 localizes to each of the spatial quality control compartments described in budding yeast, making it an effective global marker for endogenous aggregates and eliminating the need to model aggregation with ectopic proteins (Coelho et al., 2014; Kaganovich et al., 2008; Liu et al., 2010; Zhou et al., 2011). To investigate the spatial organization and dynamics of endogenous aggregates in filamentous fungi, we tagged the A. nidulans homolog of HSP104 (AN0858) with the red fluorescent protein mKate2 at its native genomic locus, and imaged live cells for 6 hr following a 45-min heat shock (45 C). One hour following heat shock, Hsp104-positive aggregates formed throughout the cell (Figure 1A), and protein expression of Hsp104 rose more than 25-fold over basal levels (Figures 1B and 1C). As recovery proceeded, these dynamic Hsp104-positive aggregates exhibited diffusive and directed motion (Movies S1 and S2), and they formed distinct clusters that yielded progressively fewer inclusions (Figures 1A and 1D). Though only a few Hsp104-positive aggregates remained after 6 hr, the intensity of these aggregates was 10-fold greater than those present earlier in recovery (Figure 1D). The protein expression levels of Hsp104 decreased as the aggregate intensities increased (compare Figures 1C and 1D), suggesting concentration of Hsp104 over time through aggregate coalescence. The condensed foci migrated with the hyphal tip during polarized growth (Movie S3), suggesting that they represent defined cytoplasmic compartments for Hsp104-mediated processing. To confirm that A. nidulans Hsp104 marks protein aggregates, we expressed fluorescently tagged (EGFP) Von Hippel-Lindau 202 Cell Reports 11, 201–209, April 14, 2015 ª2015 The Authors
(VHL) protein, which is frequently used as a model aggregating protein (Spokoini et al., 2012). Following heat shock, all Hsp104-positive inclusions colocalized with VHL (Figure S1A). We also observed a small number of Hsp104-independent VHL inclusions, which were reminiscent of the Hsp104-deficient JUNQ compartments that appeared with VHL expression in budding yeast (Figure S1A; Spokoini et al., 2012). While the majority of the condensed endogenous aggregates do not colocalize with nuclei or vacuoles, it is possible that different stress conditions may promote the formation and visualization of JUNQ- or IPOD-like (Kaganovich et al., 2008) compartments (Figures S1B and S1C). To determine how spatial organization of aggregates relates to the recovery of A. nidulans cells following heat stress, we performed a growth reinitiation assay. Transient heat shock (45 min) of A. nidulans cells yielded a temporary growth defect in which polarized tip growth stopped (Figure 1E). We determined the time at which cells reinitiated polarized growth and found that >99% of cells resumed growth within 6 hr (average time of growth reinitiation = 2.55 ± 0.04 hr; Figures 1E and 1F). Thus, the clearance of inclusions occurred within the time required for heat shock recovery, suggesting that this organization may be important for cellular fitness. Microtubules Facilitate the Association of Protein Aggregates with Hsp104 We next sought to determine whether the dynamic organization of protein aggregates after heat shock is facilitated by the cytoskeleton. Unlike budding yeast, A. nidulans uses microtubulebased transport for distribution of multiple organelles (Egan et al., 2012a; Steinberg, 2011). Thus, we hypothesized that microtubules may play a role in spatial protein quality control in A. nidulans. To evaluate the role of microtubules and filamentous actin, we pretreated cells with drugs that inhibit polymerization of each filament prior to heat shock and evaluated Hsp104 localization 30 min into recovery. Pretreatment of cells with benomyl abolished the formation Hsp104-positive aggregates (Figures 2A and 2B), suggesting that the microtubule cytoskeleton facilitates the early interaction of aggregates with Hsp104. Protein expression levels of Hsp104 in benomyl-treated cells were comparable to those of untreated cells, indicating that the decrease in aggregates was not due to differential Hsp104 expression (Figure 2C). While we cannot exclude the possibility that offtarget effects of benomyl prevented the association of protein aggregates with Hsp104 independently of microtubules, the specificity of benomyl for microtubule-mediated processes is well-established in A. nidulans (Jung et al., 1998; Sheir-Neiss et al., 1978). In contrast, filamentous actin is dispensable for the association of Hsp104 and aggregates, as pretreatment with latrunculin-A yielded Hsp104-positive inclusion numbers comparable to control experiments (Figure 2B). To better understand the movement of Hsp104-positive aggregates, we imaged live A. nidulans cells with high temporal resolution following heat shock. We analyzed motile Hsp104 particles using kymographs (Figure 2D), which are two-dimensional (2D) projections of moving particles in time-lapse movies that allowed us to quantify motile properties. Hsp104-positive aggregates underwent rapid bidirectional motility, with an average
Figure 1. Hsp104-Positive Aggregates Form, Cluster, and Coalesce during Recovery from Heat Shock (A) Time series showing the reorganization and resolution of Hsp104-positive aggregates within the same cell during a 6-hr recovery from heat shock. The fluorescent micrographs are maximumintensity projections of z series acquired at 0.5-mm intervals spanning the depth of the cell. Dashed white lines indicate the outline of the cell as traced from a differential interference contrast (DIC) projection. Scale bar, 5 mm. (B) A western blot demonstrates the induction of Hsp104 expression before heat shock (pre) and during a 6-hr recovery after heat shock. A western blot of a-tubulin expression levels is used as a loading control. (C) Quantification of Hsp104 abundance during recovery expressed as fold change of Hsp104: a-tubulin ratio over pre-heat-shock value. The mean ± SEM is shown. (D) Plot of the mean number of Hsp104-positive aggregates per cell (left axis, blue squares) and their mean fluorescence intensity (right axis, magenta squares) during a 6-hr time course following heat shock. Aggregate number and intensity were quantified from fluorescent z series of >50 cells per time point. Mean aggregate fluorescence intensities were adjusted based on Hsp104 expression levels. The mean ± SEM is shown. For mean aggregate number, the SEM at later time points was too small to resolve in the figure. (E) Cumulative frequency plot shows the time of polarized growth reinitiation in cells following recovery from heat shock (magenta line, n = 394 cells) and in non-heat-shocked cells (green line, n = 248 cells). (F) A histogram shows the temporal distribution of growth reinitiation in cells following heat shock. The magenta line represents a single Gaussian fit to the histogram data. The average time of growth reinitiation was 2.55 ± 0.04 (SEM) hr. See also Figure S1.
instantaneous velocity of 2.58 ± 1.24 mm/s (Figures 2D and 2E; Movie S2), similar to other microtubule motor-driven cargos in A. nidulans (Egan et al., 2012b; Pen˜alva, 2005; Yao et al., 2012), suggesting that microtubule-based motors may drive aggregate motility. VHL inclusions moved similarly, indicating that we were observing trafficking of protein aggregates rather than Hsp104 alone (Figure S2). Cytoplasmic Dynein Is Required for the Organization and Clearance of Hsp104-Positive Aggregates during Recovery from Heat Stress To investigate the role of microtubule-based motors in the spatial organization of protein inclusions, we simultaneously observed Hsp104-positive aggregates and the minus-end-directed motor dynein at the tips of A. nidulans cells, which contain uniformly polarized microtubules (Egan et al., 2012a; Konzack et al., 2005). We observed both plus- and minus-end-directed runs of
Hsp104-positive inclusions that colocalized with dynein (Figure 3A), suggesting an interaction with the dynein motor or its cargo. To further examine the relationship between dynein and protein aggregates, we evaluated the spatial organization of aggregates during recovery in a strain in which the endogenous dynein gene is under the control of a repressible promoter (alcA, see Table S1). The microtubule cytoskeleton is intact and polarized in the absence of dynein (Egan et al., 2012b). Genetic repression of dynein abrogated the characteristic clustering of Hsp104-positive inclusions, yielding aggregates that remained dispersed in the cytoplasm throughout the recovery process (Figure 3B). Additionally, dynein repression resulted in a dramatic increase in the number of Hsp104-positive aggregates early in recovery, indicating that dynein activity may be an important part of the initial response to protein aggregation (Figure 3C). Cell Reports 11, 201–209, April 14, 2015 ª2015 The Authors 203
Figure 2. Formation of Hsp104-Positive Aggregates following Heat Shock Requires an Intact Microtubule Cytoskeleton (A) Multicolor fluorescence micrographs showing Hsp104, nuclei, and microtubules before heat shock (top left), after heat shock (middle left), and after heat shock in cells pretreated with 2.5 mg/ml benomyl for 30 min (bottom left); and showing the Hsp104 channel only (right). Images are maximum-intensity projections of z series acquired at 0.5-mm intervals. Scale bar, 4 mm. (B) Quantification of the number of Hsp104-positive aggregates per cell before heat shock (Pre HS); after heat shock (Post HS); and after heat shock in cells pretreated with DMSO (1.2%), benomyl (Ben, 2.5 mg/ml), or latrunculin A (LatA, 12 mM). Aggregate number was quantified from fluorescent z series of >50 cells per treatment using Imaris 7.4. The mean ± SEM is shown. (C) Western blot analysis shows the detection of Hsp104 in cell lysates before heat shock (HS) and after heat shock with and without pretreatment with benomyl (Ben). (D) Kymographs generated from time-lapse sequences show the motile behavior of Hsp104-positive particles in live cells following recovery from heat shock. Images were acquired at 100-ms intervals. (E) Histogram shows the velocity distributions of motile Hsp104-positive particles. The magenta line represents a single Gaussian fit to the histogram data. The mean instantaneous velocity of Hsp104-positive particles was 2.59 ± 1.24 (SD) mm/s, n = 158. See also Figure S2.
Despite the lack of clustering and the early impediment of aggregate clearance, dynein-deficient cells ultimately cleared aggregates from the cytoplasm (Figure 3C). This may be due in part to the disaggregase activity of Hsp104, which makes direct contributions to aggregate clearance (Zhou et al., 2011). To test this hypothesis, we introduced mutations that block nucleotide hydrolysis by Hsp104 at its two ATPase sites (Hodson et al., 2012). These mutations are in the Walker B motif and, hence, we refer to this mutant as Hsp104DWB (Double Walker B). Hsp104DWB is able to bind substrates, but not disaggregate them (Hodson et al., 2012), making it a passive marker for protein aggregates. As with wild-type Hsp104, Hsp104DWB-positive aggregates appeared throughout the cytoplasm and clustered during heat shock recovery (Figure 3D). Individual aggregates within these clusters persisted beyond our 6 hr of observation, 204 Cell Reports 11, 201–209, April 14, 2015 ª2015 The Authors
yielding elevated numbers of aggregates throughout recovery (Figures S3A and S3B). This impediment of aggregate clearance was coupled with a significant delay in the reinitiation of polarized growth relative to wild-type cells (Figures S3C and S3D), further supporting the relationship between aggregate clearance and cellular fitness. Depletion of dynein in the Hsp104DWB strain caused a substantial increase in the number of aggregates that neither clustered nor were cleared from the cytoplasm (Figures 3D and 3E). To quantify the effect of dynein depletion on aggregate clustering, we determined the distribution of inclusions 1 hr into recovery by measuring the distance from every pixel located within the cytoplasm to the nearest aggregate (see the Supplemental Experimental Procedures). This provides an indication of how much aggregate-free cytoplasm surrounds each inclusion,
Figure 3. Dynein Is Required for the Organization and Clearance of Hsp104-Positive Aggregates during Recovery from Heat Stress (A) Kymographs show the motility of dynein along microtubule tracks, a subset of which colocalize with Hsp104-positive aggregates. White plus signs mark microtubule plus ends. Images were acquired in both channels simultaneously at time intervals of 100 ms. (B) Time series shows the organization and resolution of Hsp104-positive aggregates over a period of 3 hr during recovery from heat shock in a dynein-expressing cell (left) and in a cell in which dynein expression, under the control of the alcA-inducible promoter, is tightly repressed (right). (C) Plot compares the mean number of aggregates in dynein-expressing cells (cyan circles) versus dynein-deficient cells (blue squares) for 6 hr following heat shock. Aggregate number was quantified from fluorescent z series of >50 cells per time point using custom software. Different cells were imaged at each time point to avoid bleaching artifacts. The mean ± SEM is shown. (D) Time series shows the spatial organization of Hsp104DWB-positive aggregates over a 5-hr recovery from heat shock in a dynein-expressing cell (left) and in a dynein-deficient cell (right). The fluorescent micrographs are maximum-intensity projections of z series acquired at 0.5-mm intervals spanning the depth of the cell. Dashed white lines indicate the outline of the cell as traced from a DIC projection. (E). Plot compares the mean number of aggregates in dynein-expressing cells (magenta squares) versus dynein-deficient cells with Hsp104DWB (purple triangles) during a 6-hr time course following heat shock. Aggregate number was quantified from fluorescent z series of >50 cells per time point using automated methods. Different cells were imaged per time point. The mean ± SEM is shown. (F) Bar graph shows the inter-aggregate space in wild-type cells and Hsp104DWB mutants under dynein-expressing and dynein-deficient conditions 1 hr after heat shock. Inter-aggregate space was quantified from fluorescent z series of >50 cells per strain using custom software (see Experimental Procedures). The mean ± SEM is shown. See also Figure S3.
which we define as the inter-aggregate space. We found that depletion of dynein dramatically reduced the inter-aggregate space in both wild-type and Hsp104DWB cells, indicating that inclusions were more dispersed in these conditions (Figure 3F).
Aggregate clustering proceeded normally under inducing conditions (Figure S3E), indicating that the decrease in inter-aggregate space was due to differential dynein expression. Further, the 2D maximum projections used to measure inter-aggregate Cell Reports 11, 201–209, April 14, 2015 ª2015 The Authors 205
Figure 4. Persistent Hsp104DWB-Positive Aggregates Alter Endosome Trafficking (A) Fluorescence micrographs show the localization of Hsp104 (top) and Hsp104DWB (bottom) in cells grown for 20 hr at 45 C. Micrographs on the right are cropped and contrast-adjusted to highlight the presence of small puncta within the cytoplasm of the Hsp104DWB mutant. Images are maximum-intensity projections of z series acquired at 0.5-mm intervals. (B) A maximum-intensity projection of a multicolor fluorescence time-lapse sequence highlights the trajectory of early endosomes (dotted line) and Hsp104DWB-positive aggregates (top). Images were acquired in both channels simultaneously at 100-ms intervals for 10 s. Kymographs generated from time-lapse sequences are shown below ([top] the Hsp104DWB channel only; [bottom] the combined RabA and Hsp104DWB channels). (C) A histogram shows the distribution of average endosome velocities per cell for wild-type Hsp104 cells (2.53 ± 0.03 [SEM] mm/s, n = 62,268 instantaneous velocities from 48 cells) and Hsp104DWB cells (2.36 ± 0.05 [SEM] mm/s, n = 57,994 instantaneous velocities from 45 cells). (D) Bar graph shows the mean per-cell velocity of endosomes in a region confined to 10 mm from the hyphal tip, where endosome motility can be attributed to either dynein (away from the tip) or kinesin (toward the tip). The mean kinesin-driven velocity was not significantly different between wild-type Hsp104 cells and Hsp104DWB cells (2.56 ± 0.04 [SEM] mm/s versus 2.43 ± 0.05 mm/s, p = 0.0761, Mann-Whitney test), but dynein-driven endosome velocity was significantly reduced in Hsp104DWB cells (2.20 ± 0.06 mm/s versus 2.41 ± 0.05 mm/s, p = 0.0021, Mann-Whitney test). See also Figure S4.
space likely underestimated the extent of clustering, as crosssections showed unique clusters throughout the third dimension (Figure S3F). These results highlight the importance of dynein in the clustering of protein aggregates and their ultimate clearance from the cytoplasm. Hsp104DWB-Positive Aggregates Perturb Endosome Motility Given the role of dynein in aggregate clearance, we next asked if the accumulation of Hsp104-positive aggregates affects the normal trafficking of dynein cargos. To test this, we subjected cells to sustained heat stress overnight to promote aggregate accumulation. Under these conditions, Hsp104-positive inclusions were not observed in wild-type cells, indicating that steady-state Hsp104 activity is a primary agent of aggregate clearance during sustained heat stress (Figure 4A). However, in the Hsp104DWB background, prolonged heat stress produced large Hsp104DWB-positive inclusions (Figures 4A and 4B). These cells also exhibited smaller inclusions throughout the cytoplasm, suggesting that the generation and spatial organization of aggregates was constitutive under chronic heat stress (Figure 4A, inset). To evaluate the effect of these inclusions on dynein cargos, we examined the trafficking of endosomes, a known dynein cargo in 206 Cell Reports 11, 201–209, April 14, 2015 ª2015 The Authors
filamentous fungi (Egan et al., 2012a). In both wild-type and Hsp104DWB cells, endosomes were highly motile following sustained heat stress (Figure 4B). However, quantification of this motility revealed a modest but significant decrease in endosome velocity in cells containing Hsp104DWB-positive aggregates (Figure 4C). We next tested whether there was a directional component to this defect by analyzing endosome velocity in hyphal tips, where microtubules are uniformly polarized with their plus ends oriented toward the growing tips (Egan et al., 2012b; Konzack et al., 2005), allowing motility to be attributed to either dynein or its opposing motor kinesin. While we found no significant change in kinesin-driven endosome velocity, there was a subtle but significant decrease in the velocity of endosomes driven by dynein (Figure 4D). We wondered whether physical interaction between endosomes and Hsp104DWB-positive inclusions contributes to the observed decreases in velocities. To address this, we imaged endosomes and Hsp104DWB-positive inclusions simultaneously. We found that, while the majority of Hsp104DWBpositive aggregates were immotile, a subset moved and colocalized with motile endosomes (Figure 4B). Additionally, we observed possible interactions between motile endosomes and static Hsp104DWB-positive aggregates, including apparent collisions that correlated with altered endosome behavior, such as pauses and turns (Figure S4). This raises the possibility
that interactions with aggregates could contribute to the observed defects in endosome velocity. DISCUSSION In this study, we used A. nidulans to understand how filamentous fungi engage in spatial protein quality control in response to cellular stress. We found that acute heat shock promotes the formation of Hsp104-positive protein aggregates that are actively partitioned into discrete subcellular structures throughout the polarized cell. This process is dependent on microtubules, which promote both the initial formation of Hsp104-positive inclusions as well as their ultimate coalescence through the activity of dynein. Impaired aggregate clearance subtly alters the motility of microtubule-based cargos, suggesting that normal trafficking is sensitive to the accumulation of protein aggregates. In contrast to other model organisms in which damaged proteins ultimately incorporate into only one or two structures (Coelho et al., 2014; Escusa-Toret et al., 2013; Kaganovich et al., 2008; Tyedmers et al., 2010; Zhou et al., 2014), we observed numerous ultimate protein deposition sites in A. nidulans cells. We hypothesize that this could be related to both cell size and polarity. While fewer deposition sites are sufficient in relatively small and symmetrical cells such as budding yeast, fission yeast, and bacteria, longer polarized cells may need many periodic physical elements to facilitate efficient protein sequestration. Collectively, our results favor a model in which dynein facilitates aggregate clearance by promoting the assembly of Hsp104-positive inclusions into condensed compartments, effectively removing aggregates from the cytoplasm by sequestering them at sites of processing and retention. The mechanism by which dynein promotes sequestration will be an important area for future work. Our observation of Hsp104 movements that colocalize with dynein and move at speeds comparable to those driven by microtubule-based motors suggests that dynein-driven transport of aggregates may be important for their clearance. Many processes in eukaryotic cells provide potential paradigms for understanding how dynein contributes to our observed phenotype. For example, aggregate organization may resemble the assembly of stress granules, which rely on dynein for the delivery of granule components (Tsai et al., 2009). Alternatively, the mechanism could be similar to the role of dynein in transporting cargo to mammalian aggresomes, which are positioned at MTOCs (Johnston et al., 2002). While most of the condensed aggregate structures in A. nidulans did not localize to canonical MTOCs (Figure S1), non-nuclear MTOCs have been observed in A. nidulans (Konzack et al., 2005; Zekert et al., 2010), supporting the possibility that microtubule minus ends at these loci could accommodate periodic aggresome-like structures. It is also possible that dynein’s role could be to pull on microtubule-associated aggregates, resembling the positioning of nuclei in fungi (Moore and Cooper, 2010; Xiang and Fischer, 2004). Finally, our data are also consistent with dynein-dependent transport of undetected cargos such as signaling molecules, which could contribute to aggregate organization. Future work will focus on distinguishing among these possibilities.
The functional relationship between misfolded proteins and dynein led us to question of whether impaired processing of protein aggregates affects other dynein-mediated processes. Indeed, we detected small but significant velocity decreases of dynein-driven endosomes in Hsp104DWB cells following prolonged heat stress. This decrease in endosome velocity may contribute to the observed delay in growth reinitiation following heat shock, as normal polarized growth in filamentous fungi is dependent on endosome trafficking (Steinberg, 2014). Additionally, it is possible that the effects of such a subtle phenotype may manifest gradually. Such cumulative dysfunction parallels the biology of many neurodegenerative diseases, in which protein aggregation and defective microtubule-based transport are common features that may result from a lifetime of accumulated damage (Encalada and Goldstein, 2014). Future work in both neurons and model systems are needed to further illuminate the cumulative effects of subtle cellular dysfunction on trafficking. EXPERIMENTAL PROCEDURES Fungal Growth Conditions A. nidulans strains were propagated from glycerol spore stocks on agar plates of yeast extract and glucose (YG) medium (Szewczyk et al., 2006) or 1% glucose minimal medium (MM) (Nayak et al., 2010) supplemented with 1 mg/ ml uracil, 2.4 mg/ml uridine, 2.5 mg/ml riboflavin, 0.5 mg/ml pyridoxine, and 1 mg/ml para-aminobenzoic acid, depending on their auxotrophic requirements. Glufosinate extracted from Rely200 (Bayer Crop Science) was used to select for strains harboring the bar cassette (Nayak et al., 2010). AlcA(p)nudA strains were propagated on 1% fructose MM to permit sporulation. For immunoblotting, 40 ml of spore suspension at OD600 = 0.1 in YG was added to 120 3 120-mm square dishes (Hsp104 expression profile), or 25 ml of spore suspension at OD600 = 0.1 in MM was added to 100-mm diameter dishes (benomyl treatment) and incubated overnight at 25 C prior to heat shock. A. nidulans Strain Construction A. nidulans strains used in this study are listed in Table S1. DNA constructs for targeted gene deletion and tagging were made using either isothermal assembly (Gibson et al., 2009) or yeast gap repair (Orr-Weaver et al., 1983), and were integrated by homologous recombination into A. nidulans strains lacking ku70 (Nayak et al., 2006). Strains were confirmed by diagnostic PCR and fluorescence microscopy. Incorporation of the Hsp104DWB mutations were confirmed by sequencing PCR products amplified from genomic DNA. Live-Cell Imaging and Data Analysis Wide-field fluorescence images were acquired at 22 C using a custom-built DeltaVision OMX wide-field microscope (Applied Precision [GE Healthcare]; see the Supplemental Experimental Procedures). Growth Reestablishment Assay Liquid YG (3.5 ml) containing 1.75 3 105 spores was inoculated into 35-mm FluoroDishes and incubated at 25 C for 16 hr, shifted from 25 C to 45 C for 45 min, and then immediately transferred to a microscope stage (Nikon Ti Eclipse with Perfect Focus). A bright-field image was captured in a single z plane every minute for a total of 8 hr using a Plan fluor 103/0.03 objective (Nikon). Time-lapse sequences were analyzed manually in ImageJ 1.48q (NIH). Statistical Analysis and Data Presentation Statistical analysis was performed in Prism 6.0f (GraphPad), Excel version 12.3.6 (Microsoft), and MATLAB R2012a (MathWorks). Data were graphed using Prism 6.0f. Manual kymograph generation and analysis were performed using ImageJ 1.48q (NIH).
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SUPPLEMENTAL INFORMATION
Hammer, J.A., 3rd, and Sellers, J.R. (2012). Walking to work: roles for class V myosins as cargo transporters. Nat. Rev. Mol. Cell Biol. 13, 13–26.
Supplemental Information includes Supplemental Experimental Procedures, four figures, one table, and three movies and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2015.03.028.
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AUTHOR CONTRIBUTIONS M.J.E., M.A.M., and S.L.R.-P. designed and interpreted experiments. M.J.E. performed live-cell imaging. M.A.M. performed quantitative image analysis and biochemistry. H.L.E. developed automated kymograph analysis code and provided computational image analysis support. I.H.L.H. performed and analyzed growth assays and assisted with strain construction. M.J.E., M.A.M., and S.L.R.-P. wrote the paper. ACKNOWLEDGMENTS We thank the Image and Data Analysis Core and the Nikon Imaging Center at Harvard Medical School for computational image analysis and microscopy support, Gaudenz Danuser (UT Southwestern) and Applied Precision (a GE Healthcare Company) for use of the custom-built OMX V4 microscope, Xin Xiang (Uniformed Services University) for Aspergillus strains, and members of the S.L.R.P. lab for comments on the manuscript. S.L.R.P. was funded by an NIH New Innovator award (OD004268) and the Rita Allen Foundation. M.A.M. is supported by an NIH F-31 Pre-doctoral fellowship (F31NS083142). Received: November 26, 2014 Revised: February 10, 2015 Accepted: March 11, 2015 Published: April 9, 2015 REFERENCES Chin, L.S., Olzmann, J.A., and Li, L. (2010). Parkin-mediated ubiquitin signalling in aggresome formation and autophagy. Biochem. Soc. Trans. 38, 144–149. Coelho, M., Dereli, A., Haese, A., Ku¨hn, S., Malinovska, L., DeSantis, M.E., -Nørrelykke, I.M. (2013). Fission Shorter, J., Alberti, S., Gross, T., and Tolic yeast does not age under favorable conditions, but does so after stress. Curr. Biol. 23, 1844–1852. , I.M. (2014). Fusion of Coelho, M., Lade, S.J., Alberti, S., Gross, T., and Tolic protein aggregates facilitates asymmetric damage segregation. PLoS Biol. 12, e1001886. Egan, M.J., McClintock, M.A., and Reck-Peterson, S.L. (2012a). Microtubulebased transport in filamentous fungi. Curr. Opin. Microbiol. 15, 637–645.
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