FIS1 Human

What is the basic structure of human FIS1 protein?

Human FIS1 is a 16 kDa integral protein anchored in the outer mitochondrial membrane (OMM). The protein consists of two primary domains: a C-terminal transmembrane domain that anchors FIS1 to the OMM, and an N-terminal cytosolic domain. The cytosolic portion contains a bundle of six helices, with four of these helices forming two tandem tetratricopeptide repeat (TPR)-like motifs. These TPR motifs create a concave surface through their combined superhelical structure that can potentially interact with other proteins or form FIS1 dimers. Additionally, FIS1 possesses an N-terminal "arm" that can adopt different conformations and dock at the TPR motifs.

To study FIS1 structure, researchers typically employ a combination of X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations. These complementary approaches have revealed that the N-terminal arm exists in a dynamic equilibrium between "open" (OUT) and "closed" (IN) conformations, which may be critical for its biological function.

How does human FIS1 contribute to mitochondrial fission?

Human FIS1 contributes to mitochondrial fission primarily through its interaction with dynamin-related protein 1 (DRP1). While this mechanism appears conserved from yeast to humans, the precise role of human FIS1 in mitochondrial fission remains somewhat controversial. FIS1 helps regulate mitochondrial size and distribution in response to local cellular demands for ATP or calcium ions.

The molecular mechanism involves:

• FIS1 recruitment of cytosolic DRP1 to the mitochondrial outer membrane

• Formation of oligomeric DRP1 rings around mitochondria at constriction sites

• GTP hydrolysis by DRP1 providing mechanical force for membrane scission

Research methodologies to study this process include live-cell imaging with fluorescently tagged FIS1 and DRP1, co-immunoprecipitation assays to confirm protein interactions, and genetic manipulation through overexpression or knockout studies.

What is the ARCosome complex and how does FIS1 function within it?

The ARCosome is a mitochondria-endoplasmic reticulum (ER) bridging complex that includes FIS1 and plays a role in apoptotic signaling. Within this complex, FIS1 interacts with BCAP31 to form a bridge between mitochondria and the ER. This arrangement allows FIS1 to transmit proapoptotic signals from mitochondria to the ER, eventually activating procaspase-8.

To study the ARCosome, researchers employ proximity ligation assays, FRET (Förster Resonance Energy Transfer) microscopy, and subcellular fractionation techniques that can isolate mitochondria-ER contact sites. Manipulating FIS1 expression levels provides insights into how this protein regulates cross-organelle communication during cellular stress responses.

How do the conformational dynamics of the FIS1 N-terminal arm affect its function?

The N-terminal arm of human FIS1 can adopt multiple conformations, primarily characterized as "IN" (intramolecular) and "OUT" conformations. Current research indicates that these conformational states are physiologically relevant and may regulate FIS1's ability to interact with partners like DRP1.

Advanced NMR studies and molecular dynamics simulations have revealed:

• The human FIS1 arm can adopt an intramolecular conformation similar to yeast Fis1p

• This finding is supported by intrinsic tryptophan fluorescence and NMR experiments

• The FIS1 arm is sensitive to environmental changes (pH, temperature, ionic strength)

• Removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission

To investigate these dynamics, researchers use:

• Heteronuclear NOE NMR spectroscopy to study backbone dynamics on the ps-ns timescale

• Molecular dynamics simulations sampling 1000+ ns of conformational space

• Comparison of atom-atom distances between key residues in different conformational states

• Talos+ analysis of backbone torsion angles derived from chemical shifts

What analytical techniques are most effective for distinguishing between the "IN" and "OUT" conformations of FIS1?

Distinguishing between FIS1 conformational states requires multiple complementary techniques:

• NMR Chemical Shift Analysis: Comparing 1H/15N chemical shifts under different conditions (physiological pH vs. experimental conditions) reveals significant perturbations in the FIS1 arm. Kernel density plots based on secondary structural elements help visualize these differences.

• Molecular Dynamics Simulations: Starting from different initial structures (PDB ID: 1PC2 vs. homology model h1IYG), simulations over 1000 ns can reveal conformational preferences regardless of starting state. Key measurements include:

  • Cα RMSD values

  • Root mean square fluctuation (RMSF) values of arm residues

  • Sidechain atom-atom distances between residues in the arm and TPR core

• Backbone Torsion Angle Analysis: Talos+ analysis of HN, HA, CA, CB, CO, and N chemical shifts determines φ and ψ angles, which can be compared to published structures representing arm IN (PDB ID: 1IYG) and OUT (PDB ID: 1PC2) conformations.

• Heteronuclear NOE Spectroscopy: This technique detects backbone dynamics on ps-ns timescales, with values of ~0.8 for structured regions and lower values for unstructured regions, helping differentiate between arm conformational states.

How should researchers design experiments to investigate tissue-specific roles of FIS1?

Given the contradictory findings regarding FIS1's role in different cell types, researchers should consider the following experimental design:

• Cell-Type Specific Knockout Models:

  • Generate conditional knockout models using Cre-loxP systems for tissue-specific deletion

  • Compare phenotypes across multiple cell types (neuronal, hepatic, cardiac, skeletal muscle)

  • Measure mitochondrial morphology parameters (length, branching, distribution)

• Rescue Experiments:

  • Express wild-type or mutant FIS1 in knockout backgrounds

  • Test if TBC1D15 expression can rescue phenotypes in different tissues

  • Analyze complementation with orthologs from different species

• Multi-Parameter Analysis:

  • Beyond morphology, assess membrane potential, ROS production, and ATP synthesis

  • Measure mitophagy rates using mt-Keima or similar reporters

  • Evaluate integrated stress response activation through ATF4/5 targets

• Context-Dependent Studies:

  • Challenge cells with stressors (nutrient deprivation, oxidative stress)

  • Analyze temporal dynamics of FIS1 conformational changes under different conditions

  • Use live-cell imaging to track acute responses

What are the most reliable methods to quantify FIS1-DRP1 interactions in living cells?

Reliable quantification of FIS1-DRP1 interactions requires combining multiple approaches:

• Proximity-Based Detection:

  • Split fluorescent/luminescent protein complementation (BiFC, NanoBiT)

  • FRET/FLIM between tagged FIS1 and DRP1

  • Proximity ligation assay in fixed cells

  • These methods provide spatial information about interaction sites

• Biochemical Approaches:

  • Co-immunoprecipitation with antibodies against endogenous proteins

  • Crosslinking mass spectrometry to identify interaction interfaces

  • Blue native PAGE to detect native complexes

  • Importantly, interaction conditions may be sensitive to detergents and buffer compositions

• Dynamic Measurements:

  • Fluorescence recovery after photobleaching (FRAP) of GFP-DRP1 at mitochondria

  • Single-molecule tracking to measure dwell times and binding kinetics

  • Optogenetic recruitment assays to test acute FIS1-DRP1 interaction

• Controls and Validation:

  • Test arm-deleted FIS1 constructs as negative controls

  • Compare results across multiple cell types

  • Verify function using mitochondrial morphology assays in parallel

How does FIS1 contribute to metabolic disease pathology and potential therapeutic targets?

FIS1 has emerged as a key regulator of metabolic homeostasis through several mechanisms:

• Glucose Homeostasis Regulation:

  • Adenoviral-mediated acute hepatic FIS1 overexpression reduces oxidative damage and improves glucose homeostasis in high-fat diet (HFD)-fed mice

  • FIS1 activation in the liver is sufficient to restore systemic glucose homeostasis in mice fed an HFD

• Mitochondrial Stress Response Integration:

  • FIS1 serves as a signaling hub linking mitophagy and integrated stress response (ISR)

  • RNA-Seq analysis reveals that FIS1 triggers retrograde mitochondria-to-nucleus communication, upregulating ISR genes involved in:

    • Anti-oxidant defense

    • Redox homeostasis

    • Proteostasis pathways

• Inflammation Modulation:

  • FIS1-mediated ISR suppresses expression of type I interferon (IFN-I)-stimulated genes through activating transcription factor 5 (ATF5)

  • This mechanism inhibits the transactivation activity of interferon regulatory factor 3 (IRF3)

  • Metabolite analysis demonstrates that FIS1 activation leads to accumulation of fumarate, which increases ATF5 activity

Therapeutic targeting strategies could include:

• Small molecules that stabilize the FIS1-DRP1 interaction

• Compounds that modulate FIS1 arm conformational states

• Gene therapy approaches to enhance hepatic FIS1 expression in metabolic disorders

• Targeting downstream effectors in the FIS1-ATF5 axis

What experimental approaches best elucidate the role of FIS1 in neurodegenerative diseases?

To study FIS1's contribution to neurodegeneration, researchers should employ:

• Disease-Relevant Models:

  • Human iPSC-derived neurons from patients with different neurodegenerative diseases

  • Transgenic mouse models expressing disease-associated mutations

  • Primary neuronal cultures with manipulation of FIS1 expression levels

  • These models provide physiologically relevant contexts

• Mitochondrial Dynamics Assessment:

  • High-resolution live imaging of mitochondrial fission events in neurons

  • Quantification of mitochondrial transport in axons and dendrites

  • Analysis of mitochondrial quality control at synapses

  • These parameters are particularly important in highly polarized neurons

• Functional Readouts:

  • Electrophysiological measurements of neuronal activity

  • Calcium imaging at mitochondria-ER contact sites

  • Synaptic vesicle recycling assays

  • Measures of neuronal health and function beyond mitochondrial morphology

• In Vivo Approaches:

  • Neuron-specific FIS1 knockout or overexpression in mouse models

  • Behavioral assessments of motor function, learning, and memory

  • Region-specific analysis of protein aggregation and neurodegeneration

  • Correlating FIS1 dysfunction with disease progression

How can researchers reconcile conflicting data on FIS1's necessity for mitochondrial fission in different human cell types?

The literature contains contradictory findings regarding FIS1's role in mitochondrial fission, with some studies showing FIS1 knockout doesn't change mitochondrial morphology in certain cell types (e.g., HCT116 cells), while in other cells, FIS1 deletion causes mitochondrial elongation. To reconcile these contradictions:

• Systematic Comparative Analysis:

  • Standardize experimental protocols across multiple cell types

  • Perform parallel knockouts using identical CRISPR/Cas9 strategies

  • Quantify mitochondrial morphology parameters using automated, unbiased image analysis

  • This approach would reveal true cell-type differences versus technical variations

• Functional Redundancy Investigation:

  • Simultaneously knock out FIS1 and other mitochondrial receptors (MFF, MiD49, MiD51)

  • Assess compensation mechanisms that may upregulate alternative pathways

  • Perform proteomics analysis to identify differentially expressed fission factors

  • This would reveal if redundant systems mask FIS1 importance in certain contexts

• Pathway-Specific Activation:

  • Test FIS1 requirements under specific fission-inducing conditions:

    • FCCP treatment (acute mitochondrial uncoupling)

    • Hypoxia

    • Nutrient deprivation

    • Cell division

  • Different pathways may have varying dependence on FIS1

• Temporal Analysis:

  • Employ acute protein degradation systems (e.g., auxin-inducible degron)

  • Distinguish between acute versus chronic adaptation to FIS1 loss

  • Monitor compensatory changes in expression of other fission factors over time

  • This approach separates immediate effects from adaptive responses

What is the relationship between FIS1's conformational dynamics and its disputed role in human mitochondrial fission?

The dynamic conformational states of FIS1's N-terminal arm may explain its context-dependent functions:

• Conformation-Function Correlation:

  • Generate FIS1 mutants locked in specific conformational states

  • Assess their ability to recruit DRP1 and promote fission in different cell types

  • Map interaction surfaces using HDX-MS or crosslinking MS

  • This would test if different arm conformations correlate with distinct functions

• Regulatory Mechanisms:

  • Investigate post-translational modifications of the FIS1 arm

  • Determine if cellular stress triggers conformational shifts

  • Identify proteins that preferentially bind to specific FIS1 conformations

  • These factors could explain context-dependent activation

• Alternative Functions:

  • Beyond DRP1 recruitment, assess FIS1's role in:

    • Mitophagy receptor interactions

    • ARCosome formation and ER-mitochondria communication

    • Integrated stress response signaling

  • The primary function of FIS1 may vary by cell type and condition

• Evolutionary Considerations:

  • Compare human FIS1 with yeast and mouse orthologs functionally

  • Test if species-specific interaction partners explain functional differences

  • Analyze whether human cells rely more on alternative DRP1 receptors than other organisms

  • This evolutionary perspective may explain the apparently reduced importance in some human cells

What are the key technical considerations when attempting to crystallize human FIS1 for structural studies?

Crystallizing human FIS1 presents several challenges that researchers should address:

• Construct Design:

  • The dynamic N-terminal arm may hinder crystallization

  • Consider multiple constructs: full-length, arm-deleted, and arm-stabilized versions

  • The C-terminal transmembrane domain typically requires truncation

  • For membrane-bound versions, consider lipidic cubic phase crystallization

• Protein Purification Optimization:

  • Test multiple expression systems (bacterial, insect, mammalian)

  • Screen detergents carefully for membrane-containing constructs

  • Employ size-exclusion chromatography as a final purification step

  • Verify conformational homogeneity by circular dichroism before crystallization trials

• Crystallization Conditions:

  • Screen broad pH ranges (5.0-9.0) as the arm conformation is pH-sensitive

  • Test both vapor diffusion and batch crystallization methods

  • Consider co-crystallization with binding partners to stabilize conformations

  • Use surface entropy reduction mutations if initial screens fail

• Alternative Approaches:

  • Complement crystallography with solution NMR for dynamic regions

  • Consider cryo-EM for larger complexes with partners

  • Use SAXS to characterize conformational ensembles in solution

  • Integrate computational predictions with sparse experimental constraints

How can researchers accurately measure mitophagy induction by FIS1 in living cells?

Measuring FIS1-mediated mitophagy requires sensitive and specific methodologies:

• Fluorescent Reporter Systems:

  • mt-Keima: pH-sensitive fluorescent protein that changes spectral properties in lysosomes

  • mito-QC: tandem mCherry-GFP tag where GFP is quenched in lysosomes

  • TMRE labeling combined with LC3 colocalization

  • These approaches allow real-time monitoring in living cells

• Biochemical Assessments:

  • Western blotting for mitochondrial protein degradation (TOMM20, TIMM23)

  • Ubiquitination assays for mitochondrial proteins

  • Subcellular fractionation to isolate mitophagosomes

  • These provide quantitative measures of mitochondrial turnover

• Microscopy-Based Quantification:

  • Colocalization analysis of mitochondria with autophagosomes/lysosomes

  • Live-cell tracking of individual mitochondria undergoing fragmentation and clearance

  • Super-resolution microscopy to visualize mitophagy intermediates

  • These approaches provide spatial and temporal resolution

• Genetic Tools and Controls:

  • Compare wild-type FIS1 with arm-deleted mutants

  • Include positive controls (CCCP treatment) and negative controls (ATG5 knockout)

  • Test TBC1D15-dependent and independent pathways

  • These controls establish specificity for FIS1-mediated effects