| 1. |
- Henn, Arnon, et al.
(author)
-
Myosin 19 is an Outer Mitochondrial Membrane Motor and Effector of Starvation Induced Filopodia with Unique Kinetic Features
- 2016
-
In: Biophysical Journal Supplement 1. - : Elsevier BV. ; , s. 615a-616a
-
Conference paper (peer-reviewed)abstract
- The interaction between the actin cytoskeleton, myosin motors and their function in mitochondria dynamics, morphology and cellular localization is now beginning to emerge. A novel function for actin-based motors as regulators of cellular adaptations to stress, linking actin cytoskeleton remodelling to mitochondria dynamics. We reveal a novel function for myosin 19 in mitochondrial dynamics and localization during cellular response to glucose starvation. Ectopically expressed myosin 19 localizes with mitochondria at the tips of starvation-induced filopodia. Corollary to this, RNAi mediated knockdown of myosin 19 diminished their formation without evident effects on the mitochondrial network. We analyzed myosin 19 mitochondria interaction and demonstrated that it is uniquely anchored to the outer mitochondrial membrane (OMM) via a 30-residue motif, indicating that myosin 19 is a stably attached OMM molecular motor. To this end, we have purified myosin 19-3IQ motor domain construct. Myosin 19-3IQ featured characteristic actin-activated ATPase activity with moderate to slow turnover (kcat) and relatively tight KATPase. Our transient kinetics and steady state equilibrium binding experiments revealed that myosin 19-3IQ binds ATP and ADP with tight affinity that, to the best of our knowledge, have not yet been exhibited by any other myosins. We suspect that this feature allows myosin 19 to operate in a unique cellular environment that may be related to cellular stress conditions as we showed in our previous studies. The detailed knowledge of myosin 19 enzymatic adaptation will provide us with a quantitative working model of myosin 19, and will assist us to understand its cellular function. Our work reveals a novel function for myosin 19 in mitochondrial positioning during homeostasis and under stress conditions and broadens our understanding of the actin cytoskeleton- myosin -mitochondria interplay.
|
|
| 2. |
- Shneyer, Boris I., et al.
(author)
-
Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia
- 2016
-
In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 129:3, s. 543-556
-
Journal article (peer-reviewed)abstract
- Mitochondria respond to environmental cues and stress conditions. Additionally, the disruption of the mitochondrial network dynamics and its distribution is implicated in a variety of neurodegenerative diseases. Here, we reveal a new function for Myo19 in mitochondrial dynamics and localization during the cellular response to glucose starvation. Ectopically expressed Myo19 localized with mitochondria to the tips of starvation-induced filopodia. Corollary to this, RNA interference (RNAi)-mediated knockdown of Myo19 diminished filopodia formation without evident effects on the mitochondrial network. We analyzed the Myo19–mitochondria interaction, and demonstrated that Myo19 is uniquely anchored to the outer mitochondrial membrane (OMM) through a 30–45-residue motif, indicating that Myo19 is a stably attached OMM molecular motor. Our work reveals a new function for Myo19 in mitochondrial positioning under stress.
|
|
| 3. |
- Shneyer, Boris I., et al.
(author)
-
ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain
- 2017
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
-
Journal article (peer-reviewed)abstract
- The role of the actin cytoskeleton in relation to mitochondria function and dynamics is only recently beginning to be recognized. Myo19 is an actin-based motor that is bound to the outer mitochondrial membrane and promotes the localization of mitochondria to filopodia in response to glucose starvation. However, how glucose starvation induces mitochondria localization to filopodia, what are the dynamics of this process and which enzymatic adaptation allows the translocation of mitochondria to filopodia are not known. Here we show that reactive oxygen species (ROS) mimic and mediate the glucose starvation induced phenotype. In addition, time-lapse fluorescent microscopy reveals that ROS-induced Myo19 motility is a highly dynamic process which is coupled to filopodia elongation and retraction. Interestingly, Myo19 motility is inhibited by back-to-consensus-mutation of a unique residue of class XIX myosins in the motor domain. Kinetic analysis of the purified mutant Myo19 motor domain reveals that the duty ratio (time spent strongly bound to actin) is highly compromised in comparison to that of the WT motor domain, indicating that Myo19 unique motor properties are necessary to propel mitochondria to filopodia tips. In summary, our study demonstrates the contribution of actin-based motility to the mitochondrial localization to filopodia by specific cellular cues.
|
|
| 4. |
- Shneyer, Boris, et al.
(author)
-
Myosin 19 is Anchored to the Mitochondria, Affecting its Localization and Morphology
- 2015
-
In: Biophysical Journal supplement 1. - : Elsevier BV. ; , s. 303a-
-
Conference paper (peer-reviewed)abstract
- Mitochondria undergo continuous cycles of fission and fusion creating a highly dynamic network, which is essential for its proper functions in apoptosis, ATP generation and calcium homeostasis. Mitochondria long-range motility relies on the microtubule motors kinesin and dynein. Recently, actin and myosin 19 have been implicated in mitochondrial motility in vertebrates. However, the interaction of endogenous myosin 19 with the mitochondria remains unknown. Here, we show using multiple complementary approaches that endogenous myosin 19 is anchored directly to the outer mitochondrial membrane (OMM) in a monotopic fashion. We have identified a region of 30 residues at the tail domain of myosin 19, which is both essential and sufficient for myosin 19-OMM interaction. Furthermore, we have purified to near homogeneity a 45 long peptide comprised of this region to study its biochemical and biophysical properties. We performed in-vitro binding assay by fluorescence anisotropy of this specific purified peptide to vesicles with different phospholipid compositions. Our results revealed that that this peptide binds to vesicles mimicking the OMM with the highest affinity. To relate this tight binding to the mitochondria to myosin 19 ATPase activity, we have purified myosin 19-3IQ construct and measured its actin-dependent steady state ATPase activity. Interestingly, we found that it is completely inhibited by very low calcium concentration, suggesting that myosin 19 activity may be regulated by local calcium concentration. The interaction between a motor protein and an organelle, and the calcium dependence implicates that myosin 19 plays a role in mitochondria network dynamics.
|
|
| 5. |
- Ušaj, Marko, et al.
(author)
-
Kinetic adaptation of human Myo19 for active mitochondrial transport to growing filopodia tips
- 2017
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
-
Journal article (peer-reviewed)abstract
- Myosins are actin-based molecular motors which are enzymatically adapted for their cellular functions such as transportation and membrane tethering. Human Myo19 affects mitochondrial motility, and promotes their localization to stress-induced filopodia. Therefore, studying Myo19 enzymology is essential to understand how this motor may facilitate mitochondrial motility. Towards this goal, we have purified Myo19 motor domain (Myo19-3IQ) from a human-cell expression system and utilized transient kinetics to study the Myo19-3IQ ATPase cycle. We found that Myo19-3IQ exhibits noticeable conformational changes (isomerization steps) preceding both ATP and ADP binding, which may contribute to nucleotide binding regulation. Notably, the ADP isomerization step and subsequent ADP release contribute significantly to the rate-limiting step of the Myo19-3IQ ATPase cycle. Both the slow ADP isomerization and ADP release prolong the time Myo19-3IQ spend in the strong actin binding state and hence contribute to its relatively high duty ratio. However, the predicted duty ratio is lower than required to support motility as a monomer. Therefore, it may be that several Myo19 motors are required to propel mitochondria movement on actin filaments efficiently. Finally, we provide a model explaining how Myo19 translocation may be regulated by the local ATP/ADP ratio, coupled to the mitochondria presence in the filopodia.
|
|
| 6. |
- Ušaj, Marko, et al.
(author)
-
Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells
- 2018
-
In: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 558:1, s. 19-27
-
Journal article (peer-reviewed)abstract
- The myosin family of motor proteins is an attractive target of therapeutic small-molecule protein inhibitors and modulators. Milligrams of protein quantities are required to conduct proper biophysical and biochemical studies to understand myosin functions. Myosin protein expression and purification represent a critical starting point towards this goal. Established utilization of Dictyostelium discoideum, Drosophila melanogaster, insect and mouse cells for myosin expression and purification is limited, cost, labor and time inefficient particularly for (full-length) human myosins. Here we are presenting detailed protocols for production of several difficult-to-purify recombinant human myosins in efficient quantities up to 1 mg of protein per liter of cell culture. This is the first time that myosins have been purified in large scales from suspension adapted transiently and stably expressing human cells. The method is also useful for expressing other human proteins in quantities sufficient to perform extensive biochemical and biophysical characterization.
|
|
| 7. |
- Zattelman, Lilach, et al.
(author)
-
N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms
- 2017
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 292:43, s. 17804-17818
-
Journal article (peer-reviewed)abstract
- The MYO1C gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear–cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms from suspension-adapted HEK cells. MYO1CC favored the actomyosin closed state (AMC), MYO1C16 populated the actomyosin open state (AMO) and AMC equally, and MYO1C35 favored the AMO state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1CC constructs. Furthermore, global numerical simulation analysis predicted that MYO1C35 populated the actomyosin·ADP closed state (AMDC) 5-fold more than the actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC and MYO1C16 (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C35 (NTR35) docked to the X-ray structure of MYO1CC, we predicted that MYO1C35 NTR residue Arg-21 would engage in a specific interaction with post-relay helix residue Glu-469, which affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR35 peptide to MYO1CC yielded a protein that transiently mimics MYO1C35 kinetic behavior. By contrast, NTR35, which harbors the R21G mutation, was unable to confer MYO1C35-like kinetic behavior. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, adding to their kinetic diversity. We propose that this level of fine-tuning within MYO1C broadens its adaptability within cells.
|
|
| 8. |
- Zoabi, Muhammad, et al.
(author)
-
RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation
- 2014
-
In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:21, s. 13026-13038
-
Journal article (peer-reviewed)abstract
- The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.
|
|
