Hemoglobin Fibers Research Articles

High crowder concentration promotes oxygen transport function of hemoglobin S - carbon monoxide binding renders it insensitive to change in crowder concentration.

Changes in crowding conditions affect functionally important structural changes in proteins. To understand how such changes affect the function of human hemoglobin S (HbSS), the affinity of CysF9[93]β sulfhydryl group of oxy and carbonmonoxy derivatives of HbSS for 5,5’-dithiobis(2-nitrobezoate) (DTNB) were measured in buffer 5.8 < pH < 9.0 under different crowding conditions (0 – 100 g dm-3 Ficoll 70). The affinities of the two hemoglobin derivatives for DTNB decreased with increasing pH at a given crowder concentration. For oxyhemoglobin, the average instantaneous gradient of the curve resulting from the fit under each crowding condition increases with increasing crowder concentration. Presence of the crowder did not significantly affect DTNB affinity of carbonmonoxyhemoglobin. Below pH 6.7, the affinity of oxyhemoglobin for DTNB increases with increasing Ficoll 70 concentration. Conversely, above pH 6.7, the affinity of oxyHbSS for DTNB decreases with increasing Ficoll 70 concentration at a fixed pH. Crowder did not significantly affect the affinity of carbonmonoxyhemoglobin for DTNB under the pH conditions of the experiment. This insensitivity of COHbSS to presence of crowder was attributed to formation of fiber by COHbSS. Hemoglobin fiber should be insensitive to high crowder concentration. The non-responsiveness of the affinity of DTNB for COHbSS to change in activity of the medium indicates that carbon monoxide binding to the hemoglobin promotes sickling or aggregation of hemoglobin. These findings were rationalized on the basis of the greater physiological relevance of oxygen binding to hemoglobin compared to carbon monoxide binding.

Analysis of the growth of sickle hemoglobin fibers observed by DIC microscopy

Analysis of the growth of sickle hemoglobin fibers observed by DIC microscopy

Voxelotor does not inhibit sickle hemoglobin fiber formation upon complete deoxygenation

The drug voxelotor (commercially known as Oxbryta) has been approved by the US Food and Drug Administration for the treatment of sickle cell disease. It is known to reduce disease-causing sickling by inhibiting the transformation of the non-polymerizing, high-oxygen-affinity R quaternary structure of sickle hemoglobin into its polymerizing, low-affinity T quaternary structure. It has not been established whether the binding of the drug has anti-sickling effects beyond restricting the change of quaternary structure. By using a laser photolysis method that employs microscope optics, we have determined that fully deoxygenated sickle hemoglobin will assume the T structure. We show that the nucleation rates essential to generate the sickle fibers are not significantly affected by voxelotor. The method employed here should be useful for determining the mechanism of sickling inhibition for proposed drugs.

Atomic force microscopy reveals involvement of the cell envelope in biomechanical properties of sickle erythrocytes

BackgroundIntracellular hemoglobin polymerization has been supposed to be the major determinant for the elevated rigidity/stiffness of sickle erythrocytes from sickle cell anemia (SCA) patients. However, the contribution of the cell envelope remains unclear.ResultsIn this study, using atomic force microscopy (AFM), we compared the normal and sickled erythrocyte surfaces for stiffness and topography. AFM detected that sickle cells had a rougher surface and were stiffer than normal erythrocytes and that sickle cell ghosts had a rougher surface (for both outer and inner surfaces) and were thicker than normal ghosts, the latter implying a higher membrane-associated hemoglobin content/layer in the sickle cell envelope. Compared to healthy subjects, the SCA patients had lower plasma lipoprotein levels. AFM further revealed that a mild concentration of methyl-β-cyclodextrin (MβCD, a putative cholesterol-depleting reagent) could induce an increase in roughness of erythrocytes/ghosts and a decrease in thickness of ghosts for both normal and sickle cells, implying that MβCD can alter the cell envelope from outside (cholesterol in the plasma membrane) to inside (membrane-associated hemoglobin). More importantly, MβCD also caused a more significant decrease in stiffness of sickle cells than that of normal erythrocytes.ConclusionsThe data reveal that besides the cytosolic hemoglobin fibers, the cell envelope containing the membrane-associated hemoglobin also is involved in the biomechanical properties (e.g., stiffness and shape maintenance) of sickle erythrocytes.

Temperature-dependent self-assembly of biofilaments during red blood cell sickling.

Molecular self-assembly plays a vital role in various biological functions. However, when aberrant molecules self-assemble to form large aggregates, it can give rise to various diseases. For example, sickle cell disease and Alzheimer's disease are caused by self-assembled hemoglobin fibers and amyloid plaques, respectively. Here, we study the assembly kinetics of such fibers using kinetic Monte Carlo simulation. We focus on the initial lag time of these highly stochastic processes, during which self-assembly is very slow. The lag time distributions turn out to be similar for two very different regimes of polymerization, namely, (a) when polymerization is slow and depolymerization is fast and (b) the opposite case, when polymerization is fast and depolymerization is slow. Using temperature-dependent on- and off-rates for hemoglobin fiber growth, reported in recent in vitro experiments, we show that the mean lag time can exhibit non-monotonic behavior with respect to the change in temperature.

The LEST technique: Treatment of prostatic obstruction preserving antegrade ejaculation in patients with benign prostatic hyperplasia.

The objective of this study was to search for an alternative technique to relieve prostatic obstruction due to benign prostatic hyperplasia without affecting the ejaculatory function. The technical requirements are a laser with a wavelength well absorbed by water (good vaporizing effect) and at the same time by hemoglobin (good hemostatic effect) and laser fibers very resistant at high emission power allowing perfect vaporization in a contact mode. The aim of the technique is to avoid damage of the structures that allow the peripheral region of the prostate and the seminal vesicles to discharge their secretions into the posterior urethra. The orifices of the ejaculatory ducts must therefore be identified and preserved, damage of the ejaculatory ducts along their path inside the prostate must be avoided and the so called "genital sphincter" must be saved. The steps of the Leonardi Ejaculation Sparing Technique (LEST) procedure are as follow: Step 1 - ejaculatory duct orifices must be identified and the limits of the vaporization section must be marked. Step 2 - bladder neck is cleaned of the prostate hypertrophic tissue saving, as much as possible, the smooth muscle fibers of the bladder neck. Step 3 - vapo-resection of the lateral lobe (or enucleation of the adenoma) is performed. Step 4 - cautious and meticulous preparation of the prostatic apexes is obtained with saving of the orifices of the ejaculatory ducts. An antegrade ejaculation is maintained in about 80% of cases in patients without a middle lobe, although in the presence of a middle lobe this rate drops to about 50%.

Rapid and inefficient kinetics of sickle hemoglobin fiber growth.

In sickle cell disease, the aberrant assembly of hemoglobin fibers induces changes in red blood cell morphology and stiffness, which leads to downstream symptoms of the disease. Therefore, understanding of this assembly process will be important for the treatment of sickle cell disease. By performing the highest spatiotemporal resolution measurements (55 nm at 1 Hz) of single sickle hemoglobin fiber assembly to date and combining them with a model that accounts for the multistranded structure of the fibers, we show that the rates of sickle hemoglobin addition and loss have been underestimated in the literature by at least an order of magnitude. These results reveal that the sickle hemoglobin self-assembly process is very rapid and inefficient (4% efficient versus 96% efficient based on previous analyses), where net growth is the small difference between over a million addition-loss events occurring every second.

Theoretical Simulation of Red Cell Sickling Upon Deoxygenation Based on the Physical Chemistry of Sickle Hemoglobin Fiber Formation.

The polymerization of the mutant hemoglobin S upon deoxygenation to form fibers in red blood cells of patients suffering from sickle-cell anemia results in changes in cell shape and rigidity, also known as sickling, which underlie the pathology of the disease. While much has been learned about the fundamental physical chemistry of the polymerization process, transferring these insights to sickling of red cells under in vivo conditions requires being able to monitor, and ultimately predict, the time course of cellular sickling under physiological conditions of deoxygenation. To this end, we have developed an experimental technique for tracking the temporal evolution of the sickling of red blood cells under laboratory deoxygenation conditions, based on the automated analysis of sequences of microscope images and machine-learning analysis to characterize cell morphology. As an aid in the quantitative understanding of these experiments, we have developed a computational framework for simulating the time dependence of sickling in populations of red blood cells which incorporates the current theoretical and empirical understanding of the physical chemistry of the sickling process. In order to apply these techniques to our experiments, we have theoretically determined the time course of deoxygenation by solving the diffusion equation for oxygen in our experimental geometry. With this combined description, we are able to reproduce our experimentally observed kinetics of sickling, suggesting that our theoretical approach should be applicable to physiological deoxygenation scenarios.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

Modeling sickle hemoglobin fibers as one chain of coarse-grained particles

Sickle cell disease (SCD) is caused by a single point mutation in the beta-chain hemoglobin gene, resulting in the presence of abnormal hemoglobin S (HbS) in the patients' red blood cells (RBCs). In the deoxygenated state, the defective hemoglobin tetramers polymerize forming stiff fibers which distort the cell and contribute to changes in its biomechanical properties. Because the HbS fibers are essential in the formation of the sickle RBC, their material properties draw significant research interests. Here, a solvent-free coarse-grain molecular dynamics (CGMD) model is introduced to simulate single HbS fibers as a chain of particles. First, we show that the proposed model is able to efficiently simulate the mechanical behavior of single HbS fibers. Then, the zippering process between two HbS fibers is studied and the effect of depletion forces is investigated. Simulation results illustrate that depletion forces play a role comparable to direct fiber-fiber interaction via Van der Waals forces. This proposed model can greatly facilitate studies on HbS polymerization, fiber bundle and gel formation as well as interaction between HbS fiber bundles and the RBC membrane.

Predicting the morphology of sickle red blood cells using coarse-grained models of intracellular aligned hemoglobin polymers.

Sickle red blood cells (SS-RBCs) exhibit heterogeneous cell morphologies (sickle, holly leaf, granular, etc.) in the deoxygenated state due to the polymerization of the sickle hemoglobin. Experimental evidence points to a close relationship between SS-RBC morphology and intracellular aligned hemoglobin polymers. Here, we develop a coarse-grained (CG) stochastic model to represent the growth of the intracellular aligned hemoglobin polymer domain. The CG model is calibrated based on the mechanical properties (Young's modulus, bending rigidity) of the sickle hemoglobin fibers reported in experiments. The process of the cell membrane transition is simulated for physiologic aligned hemoglobin polymer configurations and mean corpuscular hemoglobin concentration. Typical SS-RBC morphologies observed in experiments can be obtained from the current model as a result of the intracellular aligned hemoglobin polymer development without introducing any further ad hoc assumptions. It is found that the final shape of SS-RBCs is primarily determined by the angular width of the aligned hemoglobin polymer domain, but it also depends, to a lesser degree, on the polymer growth rate and the cell membrane rigidity. Cell morphologies are quantified by structural shape factors, which agree well with experimental results from medical images.

Red Blood Cell Sickling During Oxygen Cycles in a Microdroplet Device

We have developed a novel microfluidic device to study repetitive sickling on individual red blood cells by replicating the physiological oxygen cycling of the vascular circulatory system (Abbyad et al., Lab Chip, 2011, 11, 813). A small number of red blood cells from sickle cell patients are encapsulated in an array of aqueous microdroplets. These microdroplets are anchored and arranged in a 2-dimensional array against the flow of the carrier oil. Precise spatial and temporal changes in oxygen concentration are obtained through gas exchange with the inert oil flowing outside the droplets. By oscillating the oxygen concentration, cycles of sickling and desickling of individual red blood cells are observed in real-time. Polarization microscopy allows for the sensitive detection of intracellular hemoglobin fibers. We observed small residual intracellular hemoglobin fibers that remain even in oxygenated conditions. Since the content of droplets in the array can be controlled, active molecules at different concentrations as well as control droplets can be measured side-by-side as they are exposed to the same environmental conditions. This was used to measure cell sickling in the presence and the absence of the anti-sickling agent glyceraldehyde. The cumulative impact of repeated sickling, such as membrane damage and cell dehydration, is believed to be central to disease pathology. We are now studying phosphatidylserine outer leaflet externalization and cell dehydration as a function of deoxygenation cycle.

A coarse-grain molecular dynamics model for sickle hemoglobin fibers

The intracellular polymerization of deoxy-sickle cell hemoglobin (HbS) has been identified as the main cause of sickle cell disease. Therefore, the material properties and biomechanical behavior of polymerized HbS fibers is a topic of intense research interest. A solvent-free coarse-grain molecular dynamics (CGMD) model is developed to represent a single hemoglobin fiber as four tightly bonded chains. A finitely extensible nonlinear elastic (FENE) potential, a bending potential, a torsional potential, a truncated Lennard-Jones potential and a Lennard-Jones potential are implemented along with the Langevin thermostat to simulate the behavior of a polymerized HbS fiber in the cytoplasm. The parameters of the potentials are identified via comparison of the simulation results to the experimentally measured values of bending and torsional rigidity of single HbS fibers. After it is shown that the proposed model is able to very efficiently simulate the mechanical behavior of single HbS fibers, it is employed in the study of the interaction between HbS fibers. It is illustrated that frustrated fibers and fibers under compression require a much larger interaction force to zipper than free fibers resulting in partial unzippering of these fibers. Continuous polymerization of the unzippered fibers via heterogeneous nucleation and additional unzippering under compression can explain the formation of HbS fiber networks and consequently the wide variety of shapes of deoxygenated sickle cells.

Sickle Hemoglobin Fiber Kinetics Revealed by Optical Pattern Generation

Sickle hemoglobin (HbS), a mutant of normal adult hemoglobin (HbA), will polymerize at concentrations above a well-defined solubility. HbS polymerization occurs by a double nucleation mechanism. A fundamental element of the mechanism is the growth of individual fibers, whose diameter (20 nm) precludes direct optical visualization. We have developed a photolytic method to measure the HbS fiber growth speed in HbS carbon monoxide derivative (COHbS) solutions. The idea of this method is that a single fiber entering a region of concentrated deoxyHbS will generate large numbers of additional fibers by heterogeneous nucleation, allowing the presence of the first fiber to be inferred even if it is not directly observed optically. We implement this method by projecting an optical pattern consisting of three parts: a large incubation circle, a small detection area, and a thin channel connecting the two. The connecting channel is turned on for just a short time; only if fiber growth is fast enough will the detection circle polymerize. Our fiber growth rates obtained from pure HbS, HbS/HbA mixtures, and partial photolysis of HbS validate a simple growth rate equation including any non-polymerizing species in the activity coefficient calculation. The monomer on-rate is determined to be 82±2 /mM/Sec. The monomer off-rate is 751±79 molecules/sec in agreement with earlier DIC observations of 850±170 molecules/sec. The method predicts a solubility of 16.0±1.1 g/dl in good agreement with 17.2 g/dl from sedimentation methods. The preceding values are for 25°C. Our measurements also rationalize the observed growth rate of the dense mass of fibers that grows more slowly along the channel and which can be visualized directly. Future uses of this method include HbS fiber bending and HbS solution fluctuations.

Real Time Monitoring of Sickle Cell Hemoglobin Fiber Formation by UV Resonance Raman Spectroscopy

In sickle cell hemoglobin, individual tetramers associate into long fibers as a consequence of the mutation at the beta6 position. In this study UV resonance Raman spectroscopy is used to monitor the formation of Hb S fibers in real time through aromatic amino acid vibrational modes. The intermolecular contact formed by the mutation site ((1)beta(1)6 Glu-->Val) of one tetramer and the (2)beta(2)85 Phe-(2)beta(2)88 Leu hydrophobic pocket on a different tetramer is observed by monitoring the increase in signal intensity of Phe vibrational modes as a function of time, yielding kinetic progress curves similar to those obtained by turbidity measurements. Comparison of individual spectra collected at early time points (<1000 s) show small Phe intensity changes, which are attributed to weak transient associations of Hb S tetramers during the initial stages of the polymerization process. At later times (1000-2000 s) Phe signal intensity steadily increases because of increasing hydrophobicity of local Phe environment, a consequence of forming more stable (1)beta(1)-(2)beta(2) contacts. Tyr and Trp vibrational modes monitor H-bond strength between critical residues at the alpha(1)beta(2) interface of individual tetramers. Kinetic progress curves generated from these signals exhibit two distinct transitions at 2040 and 7340 s. These transitions, which occur later in time than those detected either by turbidity (1560 s) or by Phe signal intensity (1680 s), are attributed to initial fiber formation and subsequent formation of larger assemblies, such as macrofibers or gels. These results provide molecular insight into the interactions governing Hb S fiber formation.

Sickle Hemoglobin Fiber Growth Rates Deduced Using Optical Channels

Sickle hemoglobin (HbS) is a point mutant of normal HbA, and will polymerize at concentrations above a well defined solubility. Polymerization occurs by a double nucleation mechanism in which homogeneous nuclei form in solution following a stochastic delay, and heterogeneous nuclei form on other polymers nucleated by either pathway. A fundamental element is the growth of individual fibers, whose diameter (20 nm) precludes direct optical visualization. Fiber growth and depolymerization have been measured by DIC microscopy, but the heterogeneous pathway makes it possible that bundles rather than single fibers have been observed, in addition to certain technical problems that make interpretation of the results less than simple. We have devised a method in which optical patterns are projected on a COHbS solution by laser photolyis, which creates deoxyHbS that can polymerize only in the illuminated area, easily allowing complex polymer structures to be created optically. In our experiment, polymers first form in an incubation circle. From the circle, a line of deoxyHbS is optically generated along which fibers can grow. Finally, a detection circle is illuminated and the connecting line is extinguished. If a polymer has entered the detection circle, thanks to heterogeneous nucleation it will fill the circle with easily observed polymers, otherwise the detection circle remains monomeric until, at some much later time, homogeneous nucleation occurs. Thus we can measure the elongation of a fiber too small to detect optically. Our preliminary results find growth rates greater than previously observed by DIC microscopy. We can also measure the growth rate of bundles by observing a mass of fibers that subsequently grow along the channel, and these grow at rates comparable to those determined by DIC microscopy. Implications of the measurements and the method will be discussed.

Study on the combination of self-assembled electrochemical active films of hemoglobin and multilayered fibers

In this study, negatively charged poly (styrenesulfonate, sodium salt) (PSS) and positively charged poly (allylamine hydrochloride) (PAH) fibers have been alternatively assembled on the polystyrene (PS) surface by using layer-by-layer (LBL) technique. Specifically ordered films have then been prepared by the self-assembling of hemoglobin (Hb) on the surface of PS/(PAH/PSS) 4/PAH or PS/(PAH/PSS) 4 fibers. Our relevant studies illustrate that compared with Hb–PS/(PAH/PSS) 4/PAH films, the self-assembled Hb–PS/(PAH/PSS) 4 films could more efficiently facilitate the direct electron transfer of Hb on glassy carbon electrode (GCE), which may be attributed to the effect of the readily electrostatic interaction between Hb and PS/(PAH/PSS) 4. Besides, cyclic voltammetry of the assembled Hb–PS/(PAH/PSS) 4 films on GCE displays a quasi-reversible electrochemical behavior in pH 5.0 acetate buffers. The average formal potential of Hb heme Fe(III)/Fe(II) couples in Hb–PS/(PAH/PSS) 4 films is observed to shift linearly between pH 3.6 and 5.7 with a slope of −52.1 mV/pH, suggesting that one proton is coupled with each electron transfer. Moreover, our results indicate that the catalytic activity to the reduction of H 2O 2 could be observed on the Hb–PS/(PAH/PSS) 4 modified GCE. A linear relationship exists between peak current and H 2O 2 concentration from 2.5 × 10 −6 to 1.2 × 10 −4 M. Consequently, our observations demonstrate that this simple and convenient strategy of the assembling Hb–PS/(PAH/PSS) 4 on GCE could effectively facilitate the direct electrochemistry of relevant heme containing proteins, which can be further utilized as a promising sensitive biosensor to detect some specific biological substrate and related biological process.

Modification of Axial Fiber Contact Residues Impact Sickle Hemoglobin Polymerization by Perturbing a Network of Coupled Interactions

The identity of intermolecular contact residues in sickle hemoglobin (HbS) fiber is largely known. However, our knowledge about combinatorial effects of two or more contact sites or the mechanistic basis of such effects is rather limited. Lys16, His20, and Glu23 of the alpha-chain occur in intra-double strand axial contacts in the sickle hemoglobin (HbS) fiber. Here we have constructed two novel double mutants, HbS (K16Q/E23Q) and (H20Q/E23Q), with a view to delineate cumulative impact of interactions emanating from the above contact sites. Far-UV and visible region CD spectra of the double mutants were similar to the native HbS indicating the presence of native-like secondary and tertiary structure in the mutants. The quaternary structures in both the mutants were also preserved as judged by the derivative UV spectra of liganded (oxy) and unliganded (deoxy) forms of the double mutants. However, the double mutants displayed interesting polymerization behavior. The polymerization behaviour of the double mutants was found to be non-additive of the individual single mutants. While HbS (H20Q/E23Q) showed inhibitory effect similar to that of HbS (E23Q), the intrinsic inhibitory propensity of the associated single mutants was totally quelled in HbS (K16Q/E23Q) double mutant. Molecular dynamics (MD) simulations studies of the isolated alpha-chains as well as a module of the fiber containing the double and associated single mutants suggested that these contact sites at the axial interface of the fiber impact HbS polymerization through a coupled interaction network. The overall results demonstrate a subtle role of dynamics and electrostatics in the polymer formation and provide insights about interaction-linkage in HbS fiber assembly.

The Kinetics of Nucleation and Growth of Sickle Cell Hemoglobin Fibers

Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times θ of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within < 1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of < 5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E A = 50 kJ mol − 1 , a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong θ( T) and weak R( T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.

Stall, Spiculate, or Run Away: The Fate of Fibers Growing towards Fluctuating Membranes

We study the dynamics of a growing semiflexible fiber approaching a membrane at an angle. At late times we find three regimes: fiber stalling, when growth stops, runaway, in which the fiber bends away from the membrane, and another regime in which spicules form. We discuss which regions of the resulting "phase diagram" are explored by (i) single and bundled actin fibers in living cells, (ii) sickle hemoglobin fibers, and (iii) microtubules inside vesicles. We complement our analysis with 3D stochastic simulations.