Mikael Björklund

Astrocytes are essential for neuronal homeostasis, and synaptic modulation and development. While astrocytes exhibit distinct Ca2+ signaling patterns, decoding their physiological roles remains challenging because conventional approaches generate nonphysiological, spatially imprecise Ca2+ surges that obscure endogenous signals. Here, we developed a tunable light-based tool to modulate astrocytic Ca2+ activity, mimicking localized spikes and global waves elicited by graded glutamate through endogenous mGluR-Gq-IP3R signaling. IP3R triple knockout abolished light-evoked Ca2+ elevations, indicating elevated Ca2+ requires ER-IP3Rs. Using this modulation platform, we found that local Ca2+ spikes enhance organelle entry into astrocytic processes, whereas global Ca2+ waves arrest organelle movement. FKBP-FRB motor recruitment assays showed that global Ca2+ elevations acutely suppress motor-driven transport when cargo adaptors are bypassed. Combining our method with astrocyte process outgrowth analysis showed that Ca2+-dependent distal organelle accumulation promotes process elongation. Together, this light-based strategy provides a versatile platform for eliciting endogenous-like astrocytic Ca2+ patterns and reveals how distinct Ca2+ patterns differentially control organelle dynamics and astrocytic structural remodeling.

BCR::ABL1 oncofusion protein drives Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ ALL), making it a critical therapeutic target. Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have revolutionized the treatment of Ph+ ALL patients. However, mutations in the kinase domain of BCR::ABL1 commonly impair the sensitivity to TKIs, resulting in drug resistance and poor prognosis in Ph+ ALL. Here we report that heat stress selectively destabilizes BCR::ABL1 and its common drug-resistant mutants without affecting the native BCR and ABL proteins through inducing liquid-to-solid phase transition. Mechanistic studies revealed that heat stress facilitated recruitment of BCR::ABL1 signaling components (e.g., SHIP2, Sts1, PI3K-p85α and Shc) in a kinase activity dependent manner and stimulated BCR::ABL1 oligomerization through its coiled-coil domain, resulting in formation of a large, thermally unstable signaling complex. This process triggers non-canonical K27-linked ubiquitination mediated by c-Cbl E3 ubiquitin ligase, ultimately leading to BCR::ABL1 degradation via the ubiquitin-proteasome pathway. Functionally, heat stress effectively suppressed proliferation of BCR::ABL1-driven leukemia cells, including drug resistant mutants in vitro and decreased tumor burden in vivo. Our findings established that thermal-based therapy as a novel strategy to selectively target and degrade both unmutated and drug-resistant BCR::ABL1 oncoproteins, offering a promising adjuvant approach to overcome TKI resistance in Ph+ ALL.

Metabolism fuels cell growth and functions. While it is well established that cellular growth rate scales with cell size, how cells alter their metabolism as they change size remains largely unexplored. Here, we conducted a systematic analysis of cell size–dependent metabolism across the NCI60 cancer cell line panel comprising a diverse range of cell sizes. We demonstrate that cellular metabolism and growth rate display 2/3 allometric scaling due to differential scaling of overall nutrient uptake and waste metabolite release with respect to cell size, with waste elimination decreasing less rapidly than nutrient uptake rate as cells grow larger. This results in cell size–dependent growth rate and predicts a maximum cell size where net nutrient uptake equals zero and cell enlargement ceases despite active metabolism. We experimentally confirm this prediction and identify that electron acceptor demand constrains cell enlargement as evidenced by depletion of intracellular aspartate and scaling of aspartate uptake, which is more than proportional to cell volume. Overall, these findings may have implications for understanding cell size homeostasis, developmental biology, and the design principles of living organisms.

Mitochondrial dysfunction is involved in numerous diseases and the aging process. The integrated stress response (ISR) serves as a critical adaptation mechanism to a variety of stresses, including those originating from mitochondria. By utilizing mass spectrometry-based cellular thermal shift assay (MS-CETSA), we uncovered that phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein (RKIP), is thermally stabilized by stresses which induce mitochondrial ISR. Depletion of PEBP1 impaired mitochondrial ISR activation by reducing eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and subsequent ISR gene expression, which was independent of PEBP1’s role in inhibiting the RAF/MEK/ERK pathway. Consistently, overexpression of PEBP1 potentiated ISR activation by heme-regulated inhibitor (HRI) kinase, the principal eIF2α kinase in the mitochondrial ISR pathway. Real-time interaction analysis using luminescence complementation in live cells revealed an interaction between PEBP1 and eIF2α, which was disrupted by eIF2α S51 phosphorylation. These findings suggest a role for PEBP1 in amplifying mitochondrial stress signals, thereby facilitating an effective cellular response to mitochondrial dysfunction. Therefore, PEBP1 may be a potential therapeutic target for diseases associated with mitochondrial dysfunction.

Mitochondrial dysfunction is involved in numerous diseases and the aging process. The integrated stress response (ISR) serves as a critical adaptation mechanism to a variety of stresses, including those originating from mitochondria. By utilizing thermal proteome profiling (TPP), we uncovered that phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein (RKIP), is thermally stabilized by stresses which induce mitochondrial ISR. Depletion of PEBP1 impaired mitochondrial ISR activation by reducing eIF2α phosphorylation and subsequent ISR gene expression, which was independent of PEBP1’s role in inhibiting the RAF/MEK/ERK pathway. Consistently, overexpression of PEBP1 potentiated ISR activation by heme-regulated inhibitor kinase (HRI), the principal eIF2α kinase in the mitochondrial ISR pathway. Real-time interaction analysis using luminescence complementation in live cells revealed an interaction between PEBP1 and eIF2α, which was disrupted by eIF2α S51 phosphorylation. These findings suggest a role for PEBP1 in amplifying mitochondrial stress signals, thereby facilitating an effective cellular response to mitochondrial dysfunction. Therefore, PEBP1 may be a potential therapeutic target for diseases associated with mitochondrial dysfunction.

Mitochondrial dysfunction is involved in numerous diseases and the aging process. The integrated stress response (ISR) serves as a critical adaptation mechanism to a variety of stresses, including those originating from mitochondria. By utilizing thermal proteome profiling (TPP), we uncovered that phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein (RKIP), is thermally stabilized by stresses which induce mitochondrial ISR. Depletion of PEBP1 impaired mitochondrial ISR activation by reducing eIF2α phosphorylation and subsequent ISR gene expression, which was independent of PEBP1's role in inhibiting the RAF/MEK/ERK pathway. Consistently, overexpression of PEBP1 potentiated ISR activation by heme-regulated inhibitor kinase (HRI), the principal eIF2α kinase in the mitochondrial ISR pathway. Real-time interaction analysis using luminescence complementation in live cells revealed an interaction between PEBP1 and eIF2α, which was disrupted by eIF2α S51 phosphorylation. These findings suggest a role for PEBP1 in amplifying mitochondrial stress signals, thereby facilitating an effective cellular response to mitochondrial dysfunction. Therefore, PEBP1 may be a potential therapeutic target for diseases associated with mitochondrial dysfunction.

Mitochondrial functions can be regulated by membrane contact sites with the endoplasmic reticulum (ER). These mitochondria–ER contact sites (MERCs) are functionally heterogeneous and maintained by various tethers. Here, we found that REEP5, an ER tubule-shaping protein, interacts with Mitofusins 1/2 to mediate mitochondrial distribution throughout the cytosol by a new transport mechanism, mitochondrial “hitchhiking” with tubular ER on microtubules. REEP5 depletion led to reduced tethering and increased perinuclear localization of mitochondria. Conversely, increasing REEP5 expression facilitated mitochondrial distribution throughout the cytoplasm. Rapamycin-induced irreversible REEP5–MFN1/2 interaction led to mitochondrial hyperfusion, implying that the dynamic release of mitochondria from tethering is necessary for normal mitochondrial distribution and dynamics. Functionally, disruption of MFN2–REEP5 interaction dynamics by forced dimerization or silencing REEP5 modulated the production of mitochondrial reactive oxygen species (ROS). Overall, our results indicate that dynamic REEP5–MFN1/2 interaction mediates cytosolic distribution and connectivity of the mitochondrial network by “hitchhiking” and this process regulates mitochondrial ROS, which is vital for multiple physiological functions.

Precise regulation of mitochondrial fusion and fission is essential for cellular activity and animal development. Imbalances between these processes can lead to fragmentation and loss of normal membrane potential in individual mitochondria. In this study, we show that MIRO-1 is stochastically elevated in individual fragmented mitochondria and is required for maintaining mitochondrial membrane potential. We further observe a higher level of membrane potential in fragmented mitochondria in fzo-1 mutants and wounded animals. Moreover, MIRO-1 interacts with VDAC-1, a crucial mitochondrial ion channel located in the outer mitochondrial membrane, and this interaction depends on the residues E473 of MIRO-1 and K163 of VDAC-1. The E473G point mutation disrupts their interaction, resulting in a reduction of the mitochondrial membrane potential. Our findings suggest that MIRO-1 regulates membrane potential and maintains mitochondrial activity and animal health by interacting with VDAC-1. This study provides insight into the mechanisms underlying the stochastic maintenance of membrane potential in fragmented mitochondria.