A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment

Tang, Xiang D.; Punch, Jesse J.; Lee, Wei-Lih

doi:10.4161/cc.8.19.9731

Cited by 49 publications

(63 citation statements)

References 45 publications

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“…We then expressed a yEGFP fusion of Num1 that lacks the PH domain (Num1ΔPH) from the endogenous NUM1 locus (Figure 1(a and b)). The PH domain is required to target the protein to the plasma membrane, and, as expected, Num1ΔPH exhibited a primarily diffuse cytosolic localization with occasional non-cortical clusters observed (Figure 1(c)) [10,14]. In contrast, in the presence of Pil1-αGFP, Num1ΔPH localized to discrete clusters at the cell cortex, indicating the protein was effectively targeted to eisosomes (Figure 1(c)).…”

Section: Resultssupporting

confidence: 53%

“…For example, mitochondria may bring Num1 molecules into closer proximity thereby increasing the density of Num1 molecules within a cluster to facilitate dynein anchoring. The number of Num1 molecules in a wildtype cluster is estimated to be ~ 14 on average [14], while the number of dynein molecules within a cortically anchored dynein focus is estimated to be ~ 6 on average [27]. Thus, it is possible that several Num1 molecules may be required to anchor a dynein dimer.…”

Section: Discussionmentioning

confidence: 99%

“…The CC domain interacts directly with the mitochondrial membrane and is also required for the Num1-dynein interaction [10–12]. The PH domain, which binds with high specificity to PI 4,5 P 2 , targets Num1 to the plasma membrane [13,14], where the protein is found in clusters as well as in a pool that is diffusely localized along the plasma membrane and with cortical ER [3,5,10,15,16]. Cluster formation, which is dependent on the CC domain and an interaction with mitochondria (Figure 1(a)), is critical for the function of Num1 in both mitochondria and dynein anchoring [10,11,17].…”

Section: Introductionmentioning

confidence: 99%

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The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.

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Section: Resultssupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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“…To estimate the number of fluorescent molecules in each spot, the measured intensity values of septin signals were compared with the intensity of mEGFP fused to a PH domain, previously shown to localize to the plasma membrane in spots containing one to two molecules of mEGFP ( Fig. 1C) (27). Based on this calibration, septin spots contained highly variable numbers of fluorescent molecules with the vast majority greater in intensity than a predicted octameric complex, which we predict should be twice the intensity of a single mEGFP (population mean PH Num1 -mEGFP = 1,974 ± 820 a.u., n = 1,510 particles; Spn1-mEGFP = 7,196 ± 4,896 a.u., n = 1,774 particles; Fig.…”

Section: Resultsmentioning

confidence: 99%

Septin assemblies form by diffusion-driven annealing on membranes

Bridges

Zhang

Mehta

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

169

223

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Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.cytoskeleton | biophysics S eptin filaments form rings, bars, and gauzes that serve as a scaffold at cell division sites; act to retract blebbed regions of membrane; and restrict diffusion between cell compartments (1-4). Septin function is required for cell division and viability in many eukaryotes whereas misregulation is associated with cancers and neurodegenerative disorders (5-8). Furthermore, septins mediate entry of both bacterial and fungal pathogens into host cells (9-11). In vivo, septin assembly is restricted both in time and in space through local activation of small GTPases such as Cdc42. Localized signaling leads to higher-order septin structures forming closely apposed to the plasma membrane at the plane of division, sites of polarity, and curved membranes (10,(12)(13)(14). Notably, eukaryotic cells of different geometries build higher-order septin assemblies of various shapes, sizes, and functions (4, 15, 16). Although septins are critical for spatial organization of cell plasma membranes, their assembly and disassembly dynamics are not understood (15).Electron microscopy (EM) studies of recombinant and immunoprecipitated Saccharomyces cerevisiae septins have shown that septins form nonpolar hetero-octameric rod-shaped complexes in high-salt buffers (>300 mM) and elongated filaments when dialyzed into low-salt buffers (<100 mM) (17, 18). Structural analyses of worm and mammalian septins have revealed that the heteromeric, rod-shaped complex is conserved (19-21). Thus, septin rods characterized to date contain two copies of each septin subunit assembled into a nonpolar, heteromeric complex (Fig. S1). Association of purified septin proteins with phosphoinositide-containing membrane monola...

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“…We created a mitochondria-cortex tether by fusing the mitochondrial targeting sequence and transmembrane domain of the mitochondrial outer membrane protein Tom70 and the pleckstrin homology (PH) domain of Num1, which previously was shown to be both necessary and sufficient for cortical targeting, to the N and C termini, respectively, of GFP (MITO-GFP-PH) (16,18). We expressed MITO-GFP-PH or constructs lacking either the mitochondria-targeting (GFP-PH) or cortex-targeting (MITO-GFP) portions in Δnum1 and fzo1-1 Δdnm1 Δnum1 cells.…”

Section: Num1 Is Essential In δFzo1 δDnm1mentioning

confidence: 99%

Endoplasmic reticulum-associated mitochondria–cortex tether functions in the distribution and inheritance of mitochondria

Lackner

Ping

Graef

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

169

284

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To elucidate the functional roles of mitochondrial dynamics in vivo, we identified genes that become essential in cells lacking the dynamin-related proteins Fzo1 and Dnm1, which are required for mitochondrial fusion and division, respectively. The screen identified Num1, a cortical protein implicated in mitochondrial division and distribution that also functions in nuclear migration. Our data indicate that Num1, together with Mdm36, forms a physical tether that robustly anchors mitochondria to the cell cortex but plays no direct role in mitochondrial division. Our analysis indicates that Num1-dependent anchoring is essential for distribution of the static mitochondrial network in fzo1 dnm1 cells. Consistently, expression of a synthetic mitochondria-cortex tether rescues the viability of fzo1 dnm1 num1 cells. We find that the cortical endoplasmic reticulum (ER) also is a constituent of the Num1 mitochondria-cortex tether, suggesting an active role for the ER in mitochondrial positioning in cells. Thus, taken together, our findings identify Num1 as a key component of a mitochondria-ER-cortex anchor, which we termed "MECA," that functions in parallel with mitochondrial dynamics to distribute and position the essential mitochondrial network.T he shape and cellular distribution of mitochondria depend on the integrated and regulated activities of mitochondrial division and fusion, motility, and tethering (1). A key question is how these mitochondrial behaviors are coordinated to shape and position mitochondria properly in response to the changing needs of the cell. To begin to address this question, an understanding of the molecular basis of mitochondrial behaviors is essential.The molecular mechanisms underlying mitochondrial division and fusion are best understood in terms of mitochondrial behaviors (1, 2). At the heart of the molecular machines that mediate mitochondrial division and fusion are dynamin-related proteins (DRPs) that function via GTP-dependent self-assembly and GTP hydrolysismediated conformational changes to remodel membranes. The DRP Dnm1/DRP1 (in yeast and mammals, respectively) drives the scission of mitochondrial membranes, and the DRPs Fzo1/ MFN1/2 and Mgm1/OPA1 mediate fusion of the outer and inner mitochondrial membranes, respectively. The relative rates of mitochondrial division and fusion are major determinants of the steadystate structure of the organelle and greatly influence its distribution. Attenuation of mitochondrial division leads to a more interconnected, collapsed, and less distributable mitochondrial network, and attenuation of mitochondrial fusion results in mitochondrial fragmentation and pronounced defects in the transmission and distribution of mtDNA.Although mitochondrial division and fusion are important, additional parallel pathways also are likely to be important for mitochondrial distribution. For example, the stable positioning of mitochondria at specific cellular locations, an indication of active tethering mechanisms, has been observed in many different cell types. In ...

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A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment

Cited by 49 publications

References 45 publications

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

Septin assemblies form by diffusion-driven annealing on membranes

Endoplasmic reticulum-associated mitochondria–cortex tether functions in the distribution and inheritance of mitochondria

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