TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes

In the paper

“under starvation conditions, autophagosomes and lysosomes move toward the cell center, facilitating the fusion between both organelles and degradation of the autophagosomal content9,10.”

What the sources say

• Partly supported

  The sections “Autophagosomes move toward the cell center” and “Significance of the rapid vectorial movement for autophagy” support movement of autophagosomes toward centrally clustered lysosomes and its role in efficient targeting. But “Anti-LC3 antibody microinjection inhibits autophagosome movement” says lysosome localization did not significantly change under starvation; the paper does not support the claim that lysosomes also move toward the cell center under those conditions. Page numbers are unavailable in our copy. (LLM, conf=0.90)

  cites [9] Kimura, S., Noda, T. & Yoshimori, T. Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes. Cell Struct. Funct. 33, 109–122 (2008). sourceExperimental — accuracy not yet measured
• Supported

  The paper states that starvation causes lysosomes to cluster near the microtubule-organizing center, facilitating fusion with autophagosomes (Abstract, page unavailable). It also describes autophagosomes moving toward that center and reports increased autolysosome formation during starvation. The paper does not say starvation itself causes autophagosomes to move inward, but the claim does not require that causal interpretation. (LLM, conf=0.90)

  cites [10] Korolchuk, V. I. et al. Lysosomal positioning coordinates cellular nutrient responses. Nat. Cell Biol. 13, 453–460 (2011). source

In the paper

“Redistribution of lysosomes toward the cell periphery is critical for cancer growth, invasion and metastasis.”

What the sources say

• ?Not checked — no full text of the sourcecites [11] Nishimura, Y., Itoh, K., Yoshioka, K., Tokuda, K. & Himeno, M. Overexpression of ROCK in human breast cancer cells: evidence that ROCK activity mediates intracellular membrane traffic of lysosomes. Pathol. Oncol. Res. 9, 83–95 (2003). source
• ?Not checked — no full text of the sourcecites [12] Macpherson, I. R. et al. CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer. J. Cell Sci. 127, 3893–3901 (2014). source
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [13] Marchesin, V. et al. ARF6-JIP3/4 regulate endosomal tubules for MT1-MMP exocytosis in cancer invasion. J. Cell Biol. 211, 339–358 (2015). source
• Partly supported

  The Introduction says peripheral lysosome trafficking facilitates protease secretion and invasion, and the Discussion links it to invasive growth and, ultimately, metastasis (pages not provided). But the Discussion distinguishes this trafficking role from Arl8b’s metabolic role in tumor growth; the paper does not establish that peripheral redistribution itself is critical for cancer growth or directly demonstrate metastasis. (LLM, conf=0.86)

  cites [14] Dykes, S. S. et al. The Arf-like GTPase Arl8b is essential for three-dimensional invasive growth of prostate cancer in vitro and xenograft formation and growth in vivo. Oncotarget 7, 31037–31052 (2016). sourceExperimental — accuracy not yet measured

In the paper

“kinesin-dependent tubulation of lysosomes is required for efficient MHCII-mediated antigen presentation in dendritic cells16–18.”

What the sources say

• ?Not checked — no full text of the sourcecites [16] Chow, A., Toomre, D., Garrett, W. & Mellman, I. Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane. Nature 418, 988–994 (2002). source
• Partly supported

  The Discussion (page unavailable) reports that endolysosomal tubules carry class II MHC molecules toward the cell surface and depend on microtubules. It says kinesin is a possible motor but that direct evidence is lacking. The paper also does not establish that tubulation is required for efficient MHCII-mediated antigen presentation. (LLM, conf=0.88)

  cites [17] Vyas, J. M. et al. Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells. J. Immunol. 178, 7199–7210 (2007). sourceExperimental — accuracy not yet measured
• Partly supported

  The paper reports in “Dynein and kinesin are required for lysosome tubulation” (page unavailable) that disrupting kinesin reduced tubular lysosomes in macrophages. Its Discussion (page unavailable) says tubular MHCII compartments in dendritic cells have been proposed to deliver antigen-loaded MHCII to immune synapses. It does not establish that kinesin-dependent tubulation is required for efficient antigen presentation in dendritic cells. (LLM, conf=0.89)

  cites [18] Mrakovic, A., Kay, J. G., Furuya, W., Brumell, J. H. & Botelho, R. J. Rab7 and Arl8 GTPases are necessary for lysosome tubulation in macrophages. Traffic 13, 1667–1679 (2012). sourceExperimental — accuracy not yet measured

In the paper

“Anterograde movement of lysosomes is regulated by the multi-subunit complex BORC, the small GTPase Arl8, and its effector SKIP, which directly interacts with the kinesin light chain, thus linking lysosomes to the plus-end-directed microtubule motor kinesin19,20.”

What the sources say

• Partly supported

  The Discussion states that Arl8 and SKIP recruit kinesin-1 to lysosomes and direct movement toward microtubule plus ends; it also says SKIP binds kinesin light chain. The cited paper does not identify BORC as a regulator of this movement (searched: BORC), so that part of the attributed claim is unsupported. (LLM, conf=0.88)

  cites [19] Rosa-Ferreira, C. & Munro, S. Ar18 and SKIP act together to link lysosomes to kinesin-1. Dev. Cell 21, 1171–1178 (2011). sourceExperimental — accuracy not yet measured
• Supported

  The Results section on Kinesin-1-dependent movement states that BORC engages the Arl8b–SKIP–Kinesin-1 pathway to regulate outward lysosome movement. The same section identifies a kinesin light-chain-binding sequence in SKIP that links lysosomes to Kinesin-1; the Discussion describes BORC recruiting Arl8b to enable that coupling. (LLM, conf=0.92)

  cites [20] Pu, J. et al. BORC, a multisubunit complex that regulates lysosome positioning. Dev. Cell 33, 176–188 (2015). source

In the paper

“The transcription factors TFEB and TFE3 exhibit the unique ability to promote expression of multiple lysosomal genes and are considered master regulators of lysosomal biogenesis2,3.”

What the sources say

• ?Not checked — no full text of the sourcecites [2] Sardiello, M. et al. A gene network regulating lysosomal biogenesis and function. Science 325, 473–477 (2009). source
• Supported

  The Results report that TFE3 increased expression of 16 of 17 lysosomal genes tested. The Discussion says TFEB induces expression of multiple lysosomal genes and identifies TFE3 as another regulator of lysosomal biogenesis; the Results also describe either factor as a potential “master” regulator, depending on its relative abundance. (LLM, conf=0.92)

  cites [3] Martina, J. A. et al. The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris. Sci. Signal. 7, ra9 (2014). source

In the paper

“LAMP-1 and LAMP-2 promote coupling of lysosomes to dynein–dynactin26,27”

What the sources say

• Partly supported

  The Distribution and motility section reports reduced dynein-mediated lysosome transport in cells lacking LAMP-1 and LAMP-2. The Discussion proposes that LAMPs help lysosomes associate with dynein–dynactin, but says the physical link is unclear; it does not establish coupling as a demonstrated mechanism. No page numbers are available in our copy. (LLM, conf=0.86)

  cites [26] Huynh, K. K. et al. LAMP proteins are required for fusion of lysosomes with phagosomes. EMBO J. 26, 313–324 (2007). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [27] Krzewski, K., Gil-Krzewska, A., Nguyen, V., Peruzzi, G. & Coligan, J. E. LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity. Blood 121, 4672–4683 (2013). source

In the paper

“We could not detect robust hydrolysis of PtdIns-4,5-P2 by either TMEM55, though we could detect hydrolysis by SHIP2, despite the fact that this enzyme has by far the weakest activity against this substrate of the 5-phosphatases44 (Supplementary Fig. 8e).”

What the sources say

• Partly supported

  The Results (page unavailable) report that SHIP2 had low activity relative to the other 5-phosphatases assayed, supporting the comparison attached to citation 44. The study tested soluble, short-chain phosphoinositide analogs and the isolated SHIP2 catalytic domain; it does not establish that intact SHIP2 has the weakest activity against native PtdIns(4,5)P2 among all 5-phosphatases. The TMEM55 observations are the citing authors’ own results, not a finding attributed to this source. (LLM, conf=0.84)

  cites [44] Tresaugues, L. et al. Structural basis for phosphoinositide substrate recognition, catalysis, and membrane interactions in human inositol polyphosphate 5-phosphatases. Structure 22, 744–755 (2014). sourceExperimental — accuracy not yet measured

In the paper

“ALG-2 interacts with the lysosomal transient receptor potential channel MCOLN1 in a calcium-dependent manner to recruit dynein–dynactin to lysosomes28,29.”

What the sources say

• Partly supported

  The Results section states that ALG-2 interacts directly with TRPML1 (MCOLN1) in a Ca²⁺-dependent manner and pulls down dynamitin, a dynactin component. It proposes that ALG-2 may link lysosomal Ca²⁺ release to dynein motors, but does not establish the stronger claim that this interaction recruits dynein–dynactin to lysosomes. (LLM, conf=0.84)

  cites [28] Li, X. et al. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation. Nat. Cell Biol. 18, 404–417 (2016). sourceExperimental — accuracy not yet measured
• Partly supported

  The Abstract states that ALG-2 binds MCOLN1 in a Ca2+-dependent manner. The Discussion proposes that ALG-2 may recruit other proteins involved in endosomal fusion or fission, but does not identify dynein–dynactin or establish its recruitment to lysosomes. Page numbers are not available in our copy. (LLM, conf=0.90)

  cites [29] Vergarajauregui, S., Martina, J. A. & Puertollano, R. Identification of the penta-EF-hand protein ALG-2 as a Ca2+-dependent interactor of mucolipin-1. J. Biol. Chem. 284, 36357–36366 (2009). sourceExperimental — accuracy not yet measured

In the paper

“Retrograde movement is regulated by Rab7 and its effector RILP. RILP interacts with the p150-glued subunit of dynactin, thus recruiting the minus-end-directed microtubule motor dynein to lysosomes23–25.”

What the sources say

• Partly supported

  The Abstract and Discussion identify RILP as a Rab7 effector involved in late endocytic transport and lysosomal organization. The Discussion only proposes that RILP might link Rab7-containing organelles to a motor complex; it does not establish that RILP binds p150-Glued or recruits dynein to lysosomes. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  cites [23] Cantalupo, G., Alifano, P., Roberti, V., Bruni, C. B. & Bucci, C. Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes. EMBO J. 20, 683–693 (2001). sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion states that Rab7 acts through RILP to recruit dynein–dynactin for minus-end transport, and that RILP directly binds dynactin’s p150Glued subunit. The paper demonstrates this recruitment on late endosomes (LEs), rather than specifically on lysosomes; it also says ORP1L and βIII spectrin are needed for transport after recruitment. (LLM, conf=0.82)

  cites [24] Johansson, M. et al. Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin. J. Cell Biol. 176, 459–471 (2007). sourceExperimental — accuracy not yet measured
• Partly supported

  The supplied passages report that Rab7-associated RILP recruits dynein–dynactin complexes to late endosomes and lysosomes, producing minus-end-directed transport. They also report recruitment of p150glued, but the cited sentence’s claim that RILP interacts with that subunit is not established: the paper says its assays revealed no direct interaction with motor-complex subunits. (LLM, conf=0.88)

  cites [25] Jordens, I. et al. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr. Biol. 11, 1680–1685 (2001). sourceExperimental — accuracy not yet measured

In the paper

“overexpression of Rab34, Rab36, Rabring7, or Rapsyn causes clustering of lysosomes in the perinuclear area.”

What the sources say

• Partly supported

  The Abstract and Figure 3 caption (page not provided) report that Rab36 overexpression clusters lysosomes around the Golgi in the perinuclear region. The Introduction describes Rab34 as regulating lysosome distribution near the Golgi, and Figure 3 compares Rab34 overexpression with Rab36. Our copy does not address Rabring7 or Rapsyn, so it does not support the full list. (LLM, conf=0.88)

  cites [30] Chen, L., Hu, J., Yun, Y. & Wang, T. Rab36 regulates the spatial distribution of late endosomes and lysosomes through a similar mechanism to Rab34. Mol. Membr. Biol. 27, 23–30 (2010). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [31] Wang, T. & Hong, W. Interorganellar regulation of lysosome positioning by the Golgi apparatus through Rab34 interaction with Rab-interacting lysosomal protein. Mol. Biol. Cell 13, 4317–4332 (2002). source
• ?Not checked — no full text of the sourcecites [32] Mizuno, K., Kitamura, A. & Sasaki, T. Rabring7, a novel Rab7 target protein with a RING finger motif. Mol. Biol. Cell 14, 3741–3752 (2003). source
• ?Not checked — no full text of the sourcecites [33] Aittaleb, M., Chen, P. J. & Akaaboune, M. Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn. J. Cell Sci. 128, 3744–3756 (2015). source

In the paper

“TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome–lysosome fusion.”

In the paper

“A modest, although statistically significant, increase in JIP4 mRNA levels was also observed in nutrient-deprived cells (Supplementary Fig. 9a).”

In the paper

“recruitment of INPP5E to the cell surface, which was used as a positive control43, induced a nearly complete loss of plasma membrane PtdIns-4,5-P2 (Supplementary Fig. 8c, d).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [43] Varnai, P., Thyagarajan, B., Rohacs, T. & Balla, T. Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells. J. Cell Biol. 175, 377–382 (2006). source

In the paper

“In agreement with this idea, we found that induction of acute stress by treatment with NaAsO2 or curcumin resulted in post-transcriptional regulation of TMEM55B, increased JIP4 and p150Glued recruitment to lysosomes, and rapid lysosomal retrograde transport via a mechanism that required JIP4 and p38 activation.”

In the paper

“Accordingly, incubation of MEFs in EBSS (starvation medium) caused significant MCOLN1 upregulation, a lysosomal protein and TFEB/TFE3 target used in this experiment as a positive control (Fig. 6e).”

In the paper

“Golgi morphology and early endosome distribution was unaffected in cells depleted of either TMEM55B or JIP4 (Supplementary Fig. 5c, d), demonstrating the specificity of TMEM55B/JIP4 in regulating late-endosome/lysosome trafficking.”

In the paper

“In cells depleted of TMEM55B the distribution significantly shifted, showing a decrease in the concentration of LAMP-1-positive structures in the perinuclear region, concomitant with a distinct increase in the number of lysosomes localized towards the cell periphery (Fig. 1g, h).”

In the paper

“In control RNAi-treated cells, lysosomes were broadly distributed throughout the cell, with a slight concentration in the perinuclear region (Fig. 1g, h).”

In the paper

“Starvation also significantly increased TMEM55B mRNA levels, and this increase was TFEB/TFE3-dependent since it was not observed in TFEB/TFE3 double knockout MEFs (Fig. 6f).”

In the paper

“Transport was exceedingly rapid, with retrograde movement detectable as early as 5 min following rapamycin induction (Supplementary Movie 1), and near complete clustering of endosomes after 30 min (Fig. 3b).”

In the paper

“we found that TMEM55B co-localized with the lysosomal marker LAMP-1 (Fig. 1b, top) but not with early endosome marker hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) (Fig. 1b, bottom).”

In the paper

“Interestingly, lysosomal clustering and JIP4 recruitment to lysosomes was prevented by incubation with p38 inhibitors, but not JNK or Erk1/2 inhibitors (Fig. 8g, h), thus revealing a novel and unexpected role of p38 in retrograde organelle transport.”

In the paper

“full length TMEM55A, as well as its N-terminal domain (TMEM55A CD), lacked the ability to bind JIP4 (Fig. 4b).”

In the paper

“JIP4-depleted cells exhibited almost complete peripheral accumulation of both lysosomes (Fig. 5a) and late endosomes (Supplementary Fig. 5c), with more than 60% of the LAMP-1 signal falling in the distal region of the cell (>15 μm from the nuclear rim) (Fig. 5b).”

In the paper

“Interestingly, lysosomal clustering and JIP4 recruitment to lysosomes was prevented by incubation with p38 inhibitors, but not JNK or Erk1/2 inhibitors (Fig. 8g, h), thus revealing a novel and unexpected role of p38 in retrograde organelle transport.”

In the paper

“Treatment of U2OS cells with NaAsO2 resulted in a clear shift in TMEM55B electrophoretic mobility, as well as an increase in TMEM55B dimerization (Fig. 8e), suggesting that TMEM55B might undergo post-translational modifications in response to acute stress.”

In the paper

“Mutation of the Cx5R motif in TMEM55B full-length did not alter the ability of this protein to induce lysosomal clustering or interact with JIP4 (Supplementary Fig. 8a, b).”

In the paper

“alanine substitutions of Leucine10 and Leucine11, residues of a putative acidic-dileucine motif at the amino termini of TMEM55B, caused accumulation of the full-length protein at the cell surface, with a minor fraction co-localizing with LAMP-1-positive structures (Supplementary Fig. 2b).”

In the paper

“GFP-TMEM55B CD exhibited a cytosolic distribution, whereas TMEM55B TM localized predominantly to the plasma membrane.”

In the paper

“As expected, TFEB and TFE3 caused a significant increase in TMEM55B mRNA and protein levels (Fig. 6a–c), thus confirming that these transcription factors modulate TMEM55B expression.”

In the paper

“JIP4 displayed no interaction with either RILP or ORP1L (Supplementary Fig. 6b)”

In the paper

“we detected binding of both proteins to endogenous JIP4 (Fig. 4b).”

In the paper

“Lysosome retrograde transport toward the immunological synapse is essential for the ability of natural killer and cytotoxic T lymphocytes to kill pathogen infected cells15”

What the sources say

• ?Not checked — no full text of the sourcecites [15] Stinchcombe, J. C., Majorovits, E., Bossi, G., Fuller, S. & Griffiths, G. M. Centrosome polarization delivers secretory granules to the immunological synapse. Nature 443, 462–465 (2006). source

In the paper

“Thirty or more cells were quantified per cell treatment and averaged to quantify the population lysosome distribution.”

In the paper

“No portion of the cell was measured more than once.”

In the paper

“In this regard, the TMEM55B/JIP4/dynein–dynactin pathway is particularly interesting, since it exhibits a dual function. It regulates the constitutive bi-directional transport of lysosomes but it also acts “on-demand” to promote retrograde lysosomal transport under certain stress conditions.”

In the paper

“Increased TMEM55B levels will favor movement of lysosomes toward the center of the cell, promoting fusion between lysosomes and autophagosomes to ensure efficient autophagic flux.”

In the paper

“Based on these results, we concluded that TMEM55B CD is both necessary and sufficient to drive retrograde trafficking of TMEM55B-targeted membranes, and does so by linking organelles to dynein motors.”

In the paper

“The resulting increase in TMEM55B levels promoted transport of lysosomes to the perinuclear region and likely facilitated lysosome/ER interactions.”

In the paper

“Amplification of the sequence of interest was normalized with a reference endogenous gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).”

In the paper

“Twenty or more cells were quantified per cell treatment and averaged to quantify the population lysosome distribution.”

In the paper

“Line scans were drawn in various regions of the cell to accurately sample total LAMP-1 distribution.”

In the paper

“Inhibition of cholesterol efflux from lysosomes in U18666A-treated cells was sensed by different cellular compartments, promoting activation of SREBF2 at the ER and TFEB/3 at the lysosomes.”

In the paper

“Thus, it was concluded that TMEM55B clusters lysosomes by recruiting the microtubule minus-end motor dynein to lysosomes, disrupting the balance of positive- and minus-end microtubule motors directing lysosomal trafficking.”

In the paper

“a tripartite complex between Rab7, FYCO1, and kinesin has also been shown to promote outward lysosomal movement21,22.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites [21] Matsuzaki, F., Shirane, M., Matsumoto, M. & Nakayama, K. I. Protrudin serves as an adaptor molecule that connects KIF5 and its cargoes in vesicular transport during process formation. Mol. Biol. Cell 22, 4602–4620 (2011). source
• ?Not checked — no full text of the sourcecites [22] Raiborg, C., Wenzel, E. M., Pedersen, N. M. & Stenmark, H. ER-endosome contact sites in endosome positioning and protrusion outgrowth. Biochem. Soc. Trans. 44, 441–446 (2016). source

In the paper

“Western blots are representative of three or more independent experiments.”

In the paper

“Therefore, our results suggest that TMEM55B does not function as a bona fide PtdIns-4,5-P2 4-phosphatase and its role in retrograde trafficking is independent of its Cx5R motif.”

In the paper

“For each measurement, blank measurements (20 µM substrate with no enzyme, as well as 250 ng protein with no substrate) were subtracted.”

In the paper

“Cell images are representative from three or more independent experiments.”

In the paper

“JIP4 is a scaffold protein that interacts with c-Jun NH2-terminal kinase (JNK) and functions as an activator of the p38 mitogen-activated protein (MAP) kinase pathway56, suggesting that JIP4 might link transport of lysosomes to signaling.”

What the sources say

• ?Not checked — no full text of the sourcecites [56] Kelkar, N., Standen, C. L. & Davis, R. J. Role of the JIP4 scaffold protein in the regulation of mitogen-activated protein kinase signaling pathways. Mol. Cell Biol. 25, 2733–2743 (2005). source

In the paper

“Experiments involving siRNA-induced knockdown of indicated proteins are representative of three or more independent experiments.”

In the paper

“Here we postulate that the interaction of TMEM55B with the JIP4–dynactin complex helps bring dynein to the lysosomal surface, thus promoting retrograde transport.”

In the paper

“Altogether, these results indicate that TMEM55B CD is required for targeting to lysosomes, and that a significant fraction of TMEM55B traffics through the plasma membrane before reaching the endo-lysosomal compartment.”

In the paper

“These results suggest that the full-length protein is required for proper localization to the endo-lysosomal compartment and that one or more endo/lysosomal targeting motifs likely reside within TMEM55B CD.”